Mario Sergio Palma

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach

BACKGROUNDnEvidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma.nnnOBJECTIVEnTo study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides.nnnMETHODSnRecombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program.nnnRESULTSnA lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6.nnnCONCLUSIONnTropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

Production of the First Effective Hyperimmune Equine Serum Antivenom against Africanized Bees

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)´2-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

Polybitoxins: a group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia paulista).

The neotropical wasp Polybia paulistinha is very aggressive and endemic in south-east Brazil, where it frequently causes stinging accidents. By using gel filtration on Sephadex G-200, followed by ion-exchange chromatography on DEAE-Cellulose under a pH gradient, a group of four toxins (designated as polybitoxins-I, II, III and IV) presenting phospholipase A2 (PLA2) activities was purified. These toxins are dimeric with mol. wts ranging from 115,000 to 132,000 and formed by different subunits. The four toxins contain very high sugar contents attached to their molecules (22-43% w/w) and presented different values of pH optimum from 7.8 to 9.0; when dissociated, only residual catalytic activities were maintained. The catalytic activities of polybitoxins (from 18 to 771 mumoles/mg per minute) are lower than that of PLA2 from Apis mellifera venom and hornetin from Vespa basalis. The polybitoxins presented a non-linear steady-state kinetic behavior for the hydrolysis of phosphatidylcholine at pH 7.9, compatible with the negative co-operativity phenomena. All of the polybitoxins were very potent direct hemolysins, especially the polybitoxins-III and IV, which are as potent as the lethal toxin from V. basalis and hornetin from Vespa flavitarsus, respectively; polybitoxin-IV presented hemolytic action 20 times higher than that of PLA2 from A. meliferra, 17 times higher than that of neutral PLA2 from Naja nigricolis and about 37 times higher than that of cardiotoxin from Naja naja atra venom.

Agelotoxin : a phospholipase A2 from the venom of the neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae)

The neotropical wasp Agelaia pallipes pallipes is aggressive and endemic in southeast of Brazil, where very often it causes stinging accidents in rural areas. By using gel filtration on Sephadex G-100, followed by high performance reversed phase chromatography in a C-18 column under acetonitrile/water gradient, the agelotoxin was purified: a toxin presenting phospholipase A(2) (PLA(2)) activity, which occurs under equilibrium of three different aggregation states: monomer (mol. wt 14 kDa), trimer (mol. wt 42 kDa) and pentamer (mol. wt 74 kDa). The enzyme presents high sugar contents attached to the protein chain (22% [w/w]) and a transition of the values of pH optimum for the substrate hydrolysis from 7.5 to 9.0, under aggregation from monomer to pentamer. All the aggregation states present Michaelian steady-state kinetic behavior and the monomer polymerization caused a decreasing of phospholipasic activity due a non-competitive inhibition promoted by the formation of a quaternary structure. The PLA(2) catalytic activity of agelotoxin changes according to its state of aggregation (from 833 to 12533 micromol mg(-1) min(-1)) and both the monomeric and oligomeric forms present lowest activities than the PLA(2) from Apis mellifera venom and hornetin from Vespa basalis. Agelotoxin is also a very potent direct hemolysin; the monomer of agelotoxin presented hemolytic actions until 200 times higher than the PbTx from P. paulista, 740 times higher than the PLA(2) from A. mellifera, 570 times higher than that of neutral PLA(2) from N. nigricolis and about 1250 times than that of cardiotoxin from Naja naja atra venom.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

The phospholipases A1 (PLA1s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2‐D, MALDI‐TOF‐TOF MS and classical protocols of protein chemistry, which included N‐ and C‐terminal sequencing. The existence of an intact form of PLA1 and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific‐IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA1, combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA2s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis.

Background Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals. Methodology and Principal Findings VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients sera. Significance Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.

Modification of the brain proteome of Africanized honeybees (Apis mellifera) exposed to a sub‐lethal doses of the insecticide fipronil

AbstractnFipronil is a phenylpyrazole insecticide that is widely used in Brazilian agriculture for pest control. Although honeybees are not targets of fipronil, studies indicate that this pesticide can be harmful to honeybees. To assess the effects of fipronil in the brain of Africanized Apis mellifera workers, this study focused on the toxico-proteome profiling of the brain of newly emerged and aged honeybee workers that were exposed to a sub-lethal dose (10xa0pg fipronil per day. i.e. 1/100 of LD50/bee/day during 5xa0days) of the insecticide. Proteomic analysis identified 25 proteins that were differentially up-regulated or down-regulated when the fipronil-exposed and non-exposed groups were compared. These proteins are potentially related to pathogen susceptibility, neuronal chemical stress, neuronal protein misfolding, and occurrence of apoptosis, ischemia, visual impairment, damaged synapse formation, brain degeneration, memory and learning impairment. The exposure of honeybees to a very low dose of fipronil, even for a short period of time (5xa0days), was sufficient to cause a series of important neuroproteomic changes in the brains of honeybees.

Peptidome profiling of venom from the social wasp Polybia paulista

Most crude venom from Polybia paulista is composed of short, linear peptides; however, only five of these peptides are structurally and functionally characterized. Therefore, the peptides in this venom were profiled using an HPLC-IT-TOF/MS and MS(n) system. The presence of type -d and -w ions that are generated from the fragmentation of the side chains was used to resolve I/L ambiguity. The distinction between K and Q residues was achieved through esterification of the α- and ε-amino groups in the peptide chains, followed by mass spectrometry analysis. Fourteen major peptides were detected in P. paulista venom and sequenced; all the peptides were synthesized on solid-phase and submitted to a series of bioassays. Five of them had been previously characterized, and nine were novel toxins. The novel peptides correspond to two wasp kinins, two chemotactic components, three mastoparans, and two peptides of unknown function. The seven novel peptides with identified functions appear to act synergistically with the previously known ones, constituting three well-known families of peptide toxins (wasp kinins, chemotactic peptides, and mastoparans) in the venom of social wasps. These multifunctional toxins can cause pain, oedema formation, haemolysis, chemotaxis of PMNLs, and mast cell degranulation in victims who are stung by wasps.

B-cell linear epitopes mapping of antigen-5 allergen from Polybia paulista wasp venom

low KIT D816V allele burden that can be detected using only a highly sensitive method, as also demonstrated by the low levels detected in our 4 cases. The analytical sensitivity of the mutation analysis performed in the present study depends on the DNA concentration of the individual samples and was typically in the range of 0.001% to 0.03%mutation-positive alleles, as previously described in detail. The 109 patients included in our study who tested negative for the KIT D816V mutation in PB were all examined clinically in our center and none had clear indications for mastocytosis based on skin examination, clinical symptoms, and s-tryptase level. Suspected elicitors among these 109 mutation-negative patients were diverse including, for example, insects, drugs, foods, exercise, and apparent idiopathic cases, and current investigations including provocation tests are ongoing in this group. A total of 29 of these patients had anaphylaxis elicited by an insect of which 5 patients lacked any skin symptoms during the acute episode whereas 22 patients had urticaria and/or angioedema and 2 patients had flushing or pruritus. A final exclusion of SM in this group would demand a BM examination, and it thus cannot be excluded that cases of SM may have been missed in this group—and especially among the patients without skin symptoms during the anaphylactic episode—because of aforementioned reasons. However, on the basis of previous studies from our group that included a group of negative controls, ie, patients with elevated s-tryptase level, in whom SM was excluded after thorough BM investigation, we find this unlikely to be the case and following this we do presently not perform a BM investigation in patients with anaphylaxis if KITD816V is not detected or UP is not found, unless other signs strongly suggestive of SM are present (ie, markedly elevated/rising basal s-tryptase level, recurring idiopathic anaphylaxis, early onset osteoporosis). In conclusion, we here present data on 4 adult patients with anaphylaxis in whom a positive KIT D816V mutation in PB subsequently led to a diagnosis of SM in spite of normal or low basal s-tryptase level and absent or inconspicuous UP skin lesions. Except for absent skin symptoms during the anaphylactic episode, the 4 patients lacked any other clinical findings that might have been a clue to the diagnosis. The use of sensitive KIT D816Vmutation analysis of PB as a diagnostic test in mastocytosis thereby clearly shows its clinical utility in patients with anaphylaxis. Further studies in a larger cohort of patients with fully characterized anaphylaxis are needed to fully interpret the value of performing the test indiscriminately in all patients with anaphylaxis as opposed to testing selected patients only on the basis of clinical or laboratory discriminators indicating mastocytosis, also given the low rate of positive results (3.5%) found in the present study. However, the method used forKITD816Vanalysis in this study has been demonstrated to have a very high diagnostic sensitivity and specificity and is furthermore quick, simple, and cheap (reagents<US

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP-III at the level of endosomal membranes from rat mast cells

50) and readily available. So, on the basis of the present findings, we find it reasonable at this point to suggest performing the test routinely in this patient group. Sigurd Broesby-Olsen, MD Athamaica Ruiz Oropeza, MD Carsten Bindslev-Jensen, MD, PhD, DMSc Hanne Vestergaard, MD, PhD Michael Boe Møller, MD, DMSc Frank Siebenhaar, MD Thomas Kristensen, PhD* Charlotte G. Mortz, MD, PhD* On behalf of theMastocytosis Centre Odense University Hospital (MastOUH) and Odense Research Centre for Anaphylaxis