Keity Souza Santos

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.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

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.

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.

Novel allergens from ancient foods: Man e 5 from manioc (Manihot esculenta Crantz) cross reacts with Hev b 5 from latex

SCOPE Manioc (Manihot esculenta) is a tuber mainly consumed in the Southern Hemisphere and used worldwide by food and chemistry industry. We aimed to recombinantly produce and characterize the first manioc allergen and evaluate its IgE reactivity in sera of Brazilian and Italian patients. METHODS AND RESULTS The molecule, termed Man e5, was expressed in E. coli, characterized by amino acid analysis, mass spectrometry, circular dichroism, HPLC, and dynamic light scattering. A tertiary structural model of the protein was produced using bioinformatics and susceptibility to pepsin digestion was analyzed in vitro. Based on its high content of charged residues, heat stability, flexibility and lack of secondary structure elements, the allergen was determined a member of the intrinsically disordered protein family. Brazilian patients were selected based on manioc allergy and Italians based on latex allergy and sensitization to Hev b 5.71% of Brazilians and 40% of Italians were in vitro IgE positive to Man e5. Cross-inhibition assays suggest a possible involvement of this allergen in the latex-fruit syndrome. CONCLUSION Man e5, the first purified allergen from manioc demonstrates IgE cross-reactivity with Hev b 5. Data suggest Hev b 5 might act as primary sensitizer and could therefore lead to allergic manifestations upon manioc consumption without prior exposition.

Proteomic analysis of urine in rats chronically exposed to fluoride

Urine is an ideal source of materials to search for potential disease‐related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2‐DE‐based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F−) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F− for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F− excretion. Urinary proteome profiles were examined using 2‐DE and Colloidal Coomassie Brilliant Blue staining. A dose‐response regarding F− intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F−, control vs. 50 ppm F− and 5 ppm F− vs. 50 ppm F− groups, respectively. Two proteins regulated by androgens (androgen‐regulated 20‐KDa protein and α‐2μ‐globulin) and one related to detoxification (aflatoxin‐B1‐aldehyde‐reductase) were identified by MALDI‐TOF‐TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F− toxicity, even in low doses.

Multiple bradykinin-related peptides from the capture web of the spider Nephila clavipes (Araneae, tetragnatidae)

Three bradykinin-related peptides (nephilakinins-I to -III) and bradykinin itself were isolated from the aqueous washing extract of the capture web of the spider Nephila clavipes by gel permeation chromatography on a Sephacryl S-100 column, followed by chromatography in a Hi-Trap Sephadex-G25 Superfine column. The novel peptides occurred in low concentrations and were sequenced through ESI-MS/MS analysis: nephilakinin-I (G-P-N-P-G-F-S-P-F-R-NH2), nephilakinin-II (E-A-P-P-G-F-S-P-F-R-NH2) and nephilakinin-III (P-S-P-P-G-F-S-P-F-R-NH2). Synthetic peptides replicated the novel bradykinin-related peptides, which were submitted to biological characterizations. Nephilakinins were shown to cause constriction on isolated rat ileum preparations and relaxation on rat duodenum muscle preparations at amounts higher than bradykinin; apparently these peptides constitute B2-type agonists of ileal and duodenal smooth muscles. All peptides including the bradykinin were moderately lethal to honeybees. These bradykinin peptides may be related to the predation of insects by the webs of N. clavipes.

Distinct Mitral Valve Proteomic Profiles in Rheumatic Heart Disease and Myxomatous Degeneration

Rheumatic heart disease (RHD) affects heart-valve tissue and is the most serious consequence of group A Streptococcus infection. Myxomatous degeneration (MXD) is the most frequent valvopathy in the western world. In the present work, key protein expression alterations in the heart-valve tissue of RHD and MXD patients were identified and characterized, with controls from cadaveric organ donors. Proteins were separated by two-dimensional (2D)-electrophoresis and identified by mass spectrometry. We found 17 differentially expressed protein spots, as compared to control samples. We observed an increased expression of ASAP-2 in the RHD patients’ valves, while collagen-VI, haptoglobin-related protein, prolargin, and cartilage oligomeric protein showed reduced expression. Valve tissue of MXD patients, on the other hand, presented lower expression of annexin-Al and A2, septin-2, SOD (Cu/Zn), and transgelin. Tissue samples from both valvopathies displayed higher expression of apolipoprotein-Al. Biglycan was downexpressed in both diseases. Vimentin and lumican showed higher expression in RHD and lower in MXD. These results suggest that key pathogenetic mechanisms are intrinsically distinct in RHD and MXD.

New manioc allergens and successful oral immunotherapy in a Brazilian allergic patient

Background Exotic tropical fruits and plants are highly consumed in Brazil and due to globalization these products are being exported worldwide. We have recently described the first manioc allergen Man e 5 that cross-reacts with Hev b 5 from latex. There are more than 70 products made up manioc starch such as drugs, soaps and fabrics and manioc allergic patients present from mild to severe reactions.

Multi-sensitization to hymenoptera venoms: diagnostic and clinical features

Background Double sensitization to both honeybee (Apis mellifera) and Yellow Jacket (Vespula ssp.) venom is common in up to 59% of Northern European Hymenoptera venom allergic patients and this rate is more than 50% in the United States. In Brazil yellow jacket is not a common wasp, but Polistes sp. and Polybia paulista poses the major risk for Brazilian patients. Reports about double sensitization involving honey bee and fire ant (Solenopsis invicta) are rare and there is nothing described about multi-sensitization to insects. Cross-reacting carbohydrate determinants (CCDs) are not present in Polistessp. venom and are not yet described for Polybia paulista neither Solenopsis invicta. Component-resolved analysis with recombinant species-specific major allergens may help to distinguish true double sensitization from crossreactivity, except for Polybia paulistaallergens for which these commercial tests are not yet available. Although there is no international consensus on whether immunotherapy regimens should generally include all venoms in multi-sensitized patients the recommendation is that immunotherapy (IT) should be extended to all venoms for which test results are positive and patients might potentially react to.