Allergie aux venins d'hyménopteres: nouveautés diagnostiques et prise en charge [Hymenoptera venom allergy: new diagnostic tools and management].

Anaphylactic reactions to hymenoptera venoms are common and, in our latitudes, mainly concern wasps and bees. Recently, molecular biology techniques have contributed to identifying and to sequencing the major allergens of insect venoms and led to the production of recombinant allergens. Assays for specific IgE directed against these recombinant allergens have recently been made available in clinical practice. They provide considerable assistance in identifying the insect responsible for an anaphylactic reaction, in particular when standard tests are positive for both wasp and bee. This article focuses on these new laboratory tests and also reviews the management of patients experiencing an anaphylactic reaction after hymenoptera sting.

Effects of mineralocorticoid and K+ concentration on K+ secretion and ROMK channel expression in a mouse cortical collecting duct cell line.

The cortical collecting duct (CCD) plays a key role in regulated K(+) secretion, which is mediated mainly through renal outer medullary K(+) (ROMK) channels located in the apical membrane. However, the mechanisms of the regulation of urinary K(+) excretion with regard to K(+) balance are not well known. We took advantage of a recently established mouse CCD cell line (mCCD(cl1)) to investigate the regulation of K(+) secretion by mineralocorticoid and K(+) concentration. We show that this cell line expresses ROMK mRNA and a barium-sensitive K(+) conductance in its apical membrane. As this conductance is sensitive to tertiapin-Q, with an apparent affinity of 6 nM, and to intracellular acidification, it is probably mediated by ROMK. Overnight exposure to 100 nM aldosterone did not significantly change the K(+) conductance, while it increased the amiloride-sensitive Na(+) transport. Overnight exposure to a high K(+) (7 mM) concentration produced a small but significant increase in the apical membrane barium-sensitive K(+) conductance. The mRNA levels of all ROMK isoforms measured by qRT-PCR were not changed by altering the basolateral K(+) concentration but were decreased by 15-45% upon treatment with aldosterone (0.3 or 300 nM for 1 and 3 h). The paradoxical response of ROMK expression to aldosterone could possibly work as a preventative mechanism to avoid excessive K(+) loss which would otherwise result from the increased electrogenic Na(+) transport and associated depolarization of the apical membrane in the CCD. In conclusion, mCCD(cl1) cells demonstrate a significant K(+) secretion, probably mediated by ROMK, which is not stimulated by aldosterone but increased by overnight exposure to a high K(+) concentration.

Medicinal application of long synthetic peptide technology.

This review covers the latest developments of long synthetic peptide technology for the rapid identification and development of malaria vaccine candidates and immunological modulators. A brief description of the two most common solid-phase synthetic procedures, together with the latest advances in optimisation of peptide chain assembly and analytical instrumentation, is given, with special attention to non-specialists. Several examples of vaccine candidates developed in the authors' or their collaborators' laboratories are also provided.

A direct role for secretory phospholipase A2 and lysophosphatidylcholine in the mediation of LPS-induced gastric injury.

Endotoxemia from sepsis can injure the gastrointestinal tract through mechanisms that have not been fully elucidated. We have shown that LPS induces an increase in gastric permeability in parallel with the luminal appearance of secretory phospholipase A2 (sPLA2) and its product, lysophosphatidylcholine (lyso-PC). We proposed that sPLA2 acted on the gastric hydrophobic barrier, composed primarily of phosphatidylcholine (PC), to degrade it and produce lyso-PC, an agent that is damaging to the mucosa. In the present study, we have tested whether lyso-PC and/or sPLA2 have direct damaging effects on the hydrophobic barriers of synthetic and mucosal surfaces. Rats were administered LPS (5 mg/kg, i.p.), and gastric contents were collected 5 h later for analysis of sPLA2 and lyso-PC content. Using these measured concentrations, direct effects of sPLA2 and lyso-PC were determined on (a) surface hydrophobicity as detected with an artificial PC surface and with intact gastric mucosa (contact angle analysis) and (b) cell membrane disruption of gastric epithelial cells (AGS). Both lyso-PC and sPLA2 increased significantly in the collected gastric juice of LPS-treated rats. Using similar concentrations to the levels in gastric juice, the contact angle of PC-coated slides declined after incubation with either pancreatic sPLA2 or lyso-PC. Similarly, gastric contact angles seen in control rats were significantly decreased in sPLA2 and lyso-PC-treated rats. In addition, we observed dose-dependent injurious effects of both lyso-PC and sPLA2 in gastric AGS cells. An LPS-induced increase in sPLA2 activity in the gastric lumen and its product, lyso-PC, are capable of directly disrupting the gastric hydrophobic layer and may contribute to gastric barrier disruption and subsequent inflammation.

BIOCHEMICAL VARIABILITY BETWEEN VENOMS FROM DIFFERENT HONEYBEE (APIS-MELLIFERA) RACES

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences.2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former.3. The polypeptides with M(r) in the range from 100,000 to 7500 da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with M(r) range from 4100 to 2000 da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.

Enzymatic variability among venoms from different subspecies of Apis mellifera (Hymenoptera: Apidae)

The enzymatic variability was analyzed in venom extracts from bees reared in different colonies of the Africanized, A. m. ligustica and A. m. carnica subspecies. The implications of this variation focused on the biochemistry differentiation and immunogenicity of these venoms. The results showed the existence of a huge variability among the subspecies as well as among the colonies for three out of the six tested components - hyaluronidase, acid phosphatase and proteases - suggesting the utilization of these features as possible biochemical markers. Furthermore, although not statistically significant, it was found that the Africanized bee venom presented slightly higher levels of protein content and esterase activity, when compared to the other subspecies. If the esterase plays a role in the pain intensity caused by the sting, as suggested elsewhere, this might suggest a reason for a bigger algogenicity of this venom in relation to that of European bees. On the other hand, A. m. ligustica bees presented the highest levels of proteolytic and acid phosphatase activities, whose functions are not enlightened in Hymenoptera venoms. The A. m. carnica workers presented the highest hyaluronidase and the lowest acid phosphatase activity levels. The extremely variable results among colonies of the same subspecies and among subspecies, for the tested venom components, justify the absence of correlation between allergic reactions and tests with pooled venom.

Chemometric analysis of Hymenoptera toxins and defensins: A model for predicting the biological activity of novel peptides from venoms and hemolymph

When searching for prospective novel peptides, it is difficult to determine the biological activity of a peptide based only on its sequence. The trial and error approach is generally laborious, expensive and time consuming due to the large number of different experimental setups required to cover a reasonable number of biological assays. To simulate a virtual model for Hymenoptera insects, 166 peptides were selected from the venoms and hemolymphs of wasps, bees and ants and applied to a mathematical model of multivariate analysis, with nine different chemometric components: GRAVY, aliphaticity index, number of disulfide bonds, total residues, net charge, pI value, Boman index, percentage of alpha helix, and flexibility prediction. Principal component analysis (PCA) with non-linear iterative projections by alternating least-squares (NIPALS) algorithm was performed, without including any information about the biological activity of the peptides. This analysis permitted the grouping of peptides in a way that strongly correlated to the biological function of the peptides. Six different groupings were observed, which seemed to correspond to the following groups: chemotactic peptides, mastoparans, tachykinins, kinins, antibiotic peptides, and a group of long peptides with one or two disulfide bonds and with biological activities that are not yet clearly defined. The partial overlap between the mastoparans group and the chemotactic peptides, tachykinins, kinins and antibiotic peptides in the PCA score plot may be used to explain the frequent reports in the literature about the multifunctionality of some of these peptides. The mathematical model used in the present investigation can be used to predict the biological activities of novel peptides in this system, and it may also be easily applied to other biological systems. © 2011 Elsevier Inc.

Proteome and phosphoproteome of Africanized and European honeybee venoms

Honey bee venom toxins trigger immunological, physiological, and neurological responses within victims. The high occurrence of bee attacks involving potentially fatal toxic and allergic reactions in humans and the prospect of developing novel pharmaceuticals make honey bee venom an attractive target for proteomic studies. Using label-free quantification, we compared the proteome and phosphoproteome of the venom of Africanized honeybees with that of two European subspecies, namely Apis mellifera ligustica and A. m. carnica. From the total of 51 proteins, 42 were common to all three subspecies. Remarkably, the toxins melittin and icarapin were phosphorylated. In all venoms, icarapin was phosphorylated at the 205Ser residue, which is located in close proximity to its known antigenic site. Melittin, the major toxin of honeybee venoms, was phosphorylated in all venoms at the 10Thr and 18Ser residues. 18Ser phosphorylated melittin-the major of its two phosphorylated forms-was less toxic compared to the native peptide. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

BIOCHEMICAL VARIABILITY BETWEEN VENOMS FROM DIFFERENT HONEYBEE (APIS-MELLIFERA) RACES

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences.2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former.3. The polypeptides with M(r) in the range from 100,000 to 7500 da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with M(r) range from 4100 to 2000 da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.

The mitochondrial genome of the stingless bee Melipona bicolor (Hymenoptera, Apidae, Meliponini): sequence, gene organization and a unique tRNA translocation event conserved across the tribe Meliponini

At present a complete mtDNA sequence has been reported for only two hymenopterans, the Old World honey bee, Apis mellifera and the sawfly Perga condei. Among the bee group, the tribe Meliponini (stingless bees) has some distinction due to its Pantropical distribution, great number of species and large importance as main pollinators in several ecosystems, including the Brazilian rain forest. However few molecular studies have been conducted on this group of bees and few sequence data from mitochondrial genomes have been described. In this project, we PCR amplified and sequenced 78% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was partially sequenced). We also report the genome organization (gene content and order), gene translation, genetic code, and other molecular features, such as base frequencies, codon usage, gene initiation and termination. We compare these characteristics of M. bicolor to those of the mitochondrial genome of A. mellifera and other insects. A highly biased A+T content is a typical characteristic of the A. mellifera mitochondrial genome and it was even more extreme in that of M. bicolor. Length and compositional differences between M. bicolor and A. mellifera genes were detected and the gene order was compared. Eleven tRNA gene translocations were observed between these two species. This latter finding was surprising, considering the taxonomic proximity of these two bee tribes. The tRNA Lys gene translocation was investigated within Meliponini and showed high conservation across the Pantropical range of the tribe.

Notes on oil sources for the bee genus Caenonomada(Hymenoptera, Apidae, Tapinotaspidini)

It is reported for the first time oil collecting by bees of the genus Caenonomada on flowers of Plantaginaceae. Females of Caenonomada unicalcarata were observed collecting oil on flowers of Angelonia cornigera, and males and females of Caenonomada bruneri and C. aff. unicalcarata were observed on flowers of Angelonia and Monopera (Plantaginaceae). The record of Caenonomada on Plantaginaceae suggests the use of trichomatic oil glands as a primitive condition in the tribe Tapinotaspidini.

A scientific note about spectroscopic analysis of honey bee brood comb cappings in hygienic and non-hygienic honey bee colonies

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Phoretic mites (Acari: Chaetodactylidae) associated with the solitary bee Tetrapedia diversipes (Apidae: Tetrapediini)

Solitary bees of the genus Tetrapedia have a specific association with mites of the genus Roubikia (Chaetodactylidae). These mites are frequently found attached to active Tetrapedia bees. We quantified the number of mites on individuals of Tetrapedia diversipes Klug and examined the interaction between these species. Nests of T. diversipes were obtained from trap-nests placed in four localities in Sao Paulo, Brazil. The study lasted from March 2007 to February 2009. Out of a total of 650 nests with emergences, 118 were infested with mites (Roubikia sp.). From these nests, 176 individuals of T. diversipes emerged with mites on their bodies. Additionally, six individuals of Coelioxoides waltheriae, the specific kleptoparasitic bee to T. diversipes, emerged. Mites were attached mainly to the mesosoma. All nests infected with mites did not presented mortality of the immature. The mortality rate of nests was inversely related to the level of mite infestation, suggesting a mutualistic interaction in which mites may remove fungi from the nests, while the bees would provide the mites with transport, dispersal, and shelter.

Pollen sources of the orchid bee Euglossa annectans Dressler 1982 (Hymenoptera: Apidae, Euglossini) analyzed from larval provisions

In order to analyze the pollen resources used by the orchid bee Euglossa annectans, samples of larval provisions from cells under construction were taken from 12 different trap nests (wooden boxes) on Santa Catarina Island, southern Brazil. The 43 samples collected between 2002 and 2005 represented all months except December. Overall, 74 pollen types from 24 families were distinguished. Among the 26 pollen types that reached more than 10% in monthly means, the families Melastomataceae, Bromeliaceae, Ochnaceae, Fabaceae, and Myrtaceae were most frequently represented. The Shannon-Weaver diversity index H' for the 43 brood cells varied from 0.10-1.65 and the annual diversity was 0.98. Similarity indices ranged from 0 to 0.87 and were highest during spring and summer. The results characterize E. annectans as a polylectic species. Based on these data, we can conclude that Euglossa females may act as pollinators of many forest species.

Cuticular hydrocarbons in the stingless bee Schwarziana quadripunctata (Hymenoptera, Apidae, Meliponini): differences between colonies, castes and age

Chemical communication is of fundamental importance to maintain the integration of insect colonies. In honey bees, cuticular lipids differ in their composition between queens, workers and drones. Little is known, however, about cuticular hydrocarbons in stingless bees. We investigated chemical differences in cuticular hydrocarbons between different colonies, castes and individuals of different ages in Schwarziana quadripunctata. The epicuticle of the bees was extracted using the nonpolar solvent hexane, and was analyzed by means of a gas chromatograph coupled with a mass spectrometer. The identified compounds were alkanes, branched-alkanes and alkenes with chains of 19 to 33 carbon atoms. Discriminant analyses showed clear differences between all the groups analyzed. There were significant differences between bees from different colonies, workers of different age and between workers and virgin queens.