Roberto H. P. Moraes

Enhancement of Sf‐9 Cell Growth and Longevity through Supplementation of Culture Medium with Hemolymph

The benefits of insect cell culture medium supplementation with hemolymph of Lonomia obliqua were investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3‐fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti‐apoptotic factor, a growth‐promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supplementation of the culture medium with hemolymph.

Purification and Characterization of an Anti-Apoptotic Protein Isolated from Lonomia obliqua Hemolymph

Previously it was reported that supplementation of insect cell culture with Lonomia obliquahemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58–63). In this work the anti‐apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf‐9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti‐apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf‐9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliquahemolymph is due to the presence of potent anti‐apoptotic factors.

Description of an injury in a human caused by a false tocandira (Dinoponera gigantea, Perty, 1833) with a revision on folkloric, pharmacological and clinical aspects of the giant ants of the genera Paraponera and Dinoponera (sub-family Ponerinae)

The authors observed an injury caused by the sting of a false tocandira ant in the hand of an amateur fisherman and they describe the clinical findings and the evolution of the envenoming, which presented an acute and violent pain, cold sweating, nausea, a vomiting episode, malaise, tachycardia and left axillarys lymphadenopathy. About three hours after the accident, still feeling intense pain in the place of the sting, he presented an episode of great amount of blood in the feces with no history of digestive, hematological or vascular problems. The intense pain decreased after eight hours, but the place stayed moderately painful for about 24 hours. In that moment, he presented small grade of local edema and erythema. The authors still present the folkloric, pharmacological and clinical aspects related to the tocandiras stings, a very interesting family of ants, which presents the largest and more venomous ants of the world.

Antiviral activity of the hemolymph of Lonomia obliqua (Lepidoptera: Saturniidae)

Potent antiviral activity against measles, influenza and polio viruses was observed in the hemolymph of Lonomia obliqua. The antiviral protein responsible for this activity was isolated, purified by gel filtration chromatography using a gel filtration column system (Superdex 75) and further fractionated using a Resource-Q ion exchange column system. Experiments with the purified protein led to a 157-fold reduction (from 3.3+/-1.25 x 10(7) to 2.1+/-1.5 x 10(5) TCID(50)mL(-1)) in measles virus production and a 61-fold reduction (from 2.8+/-1.08 x 10(9) to 4.58+/-1.42 x 10(7)mL(-1)) in polio virus production. Heating and freezing seem to have no influence over its antiviral activity. Also, the protein does not display virucidal activity and does not act on receptors on the cell membrane. The observations suggest an intracellular mechanism of action and that the protein may act as a constitutive agent that affects the innate antiviral immune response.

Surtos epidêmicos de dermatite causada por mariposas do gênero Hylesia (Lepidóptera: Hemileucidae) no Estado de São Paulo, Brasil

Epidemic outbreaks of dermatitis caused by Hylesia sp wich occurred in the coastal region of the State of S. Paulo during the period of December 1989 to December 1991 are confirmed. The incident assumed its greatest intensity in Bertioga, in Santos County, where 612 cases were registered. The outbreak also affected 12 other counties in that region and it was estimated that hundred of affected persons sought the Local Health Service Care. The majority presented with erythematous and prutiginous lesions and papula which lasted 7 to 14 days on average. Treatment consisted of systemic administration of antihistamines, and the use of topic corticosteroides and cold compresses. During the abovementioned period, three epidemic episodes, coninciding with the rainy season (November to January), ocurred. They were first observed in the northern part of the coastal area and then spread to the south. Information as to how avoid contact with the moth was the main prophylactic measure. In highly infested buildings the effectiveness of residual insecticide procedure was used in the attempt to reduce the level of moth infestation. Satisfatory results were obtained with deltametrin applied in a dosage of 50 mg/m2 of wall.Epidemic outbreaks of dermatitis caused by Hylesia sp which occurred in the coastal region of the State of S. Paulo during the period of December 1989 to December 1991 are confirmed. The incident assumed its greatest intensity in Bertioga, in Santos County, where 612 cases were registered. The outbreak also affected 12 other counties in that region and it was estimated that hundred of affected persons sought the Local Health Service Care. The majority presented with erythematous and prutiginous lesions and papula which lasted 7 to 14 days on average. Treatment consisted of systemic administration of antihistamines, and the use of topic corticosteroids and cold compresses. During the above-mentioned period, three epidemic episodes, coinciding with the rainy season (November to January), occurred. They were first observed in the northern part of the coastal area and then spread to the south. Information as to how avoid contact with the moth was the main prophylactic measure. In highly infested buildings the effectiveness of residual insecticide procedure was used in the attempt to reduce the level of moth infestation. Satisfactory results were obtained with deltametrin applied in a dosage of 50 mg/m2 of wall.

Expression of an antiviral protein from Lonomia obliqua hemolymph in baculovirus/insect cell system

Abstract The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.

Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of Lonomia obliqua

Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system.

Identification and characterization of a baculovirus from Lonomia obliqua (Lepidoptera: Saturniidae).

A baculovirus has been isolated from larvae of Lonomia obliqua, a Saturniidae of medical importance due to a potent toxin found in their spines. Electron Microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra of approximately 1 microm in diameter containing multiple nucleocapsids per envelope. This baculovirus was thus named Lonomia obliqua multicapsid nucleopolyhedrovirus (LoobMNPV). Restriction endonuclease profiles of viral DNA digested with three restriction enzymes were obtained and the genome size was estimated to be 95.52 +/- 2.3 kbp. The polyhedrin gene of LoobMNPV was identified and its DNA sequence was determined. Phylogenetic analysis of the polyhedrin gene showed that the LoobMNPV polyhedrin belongs to group I NPV and that it is closely related to the polyhedrin of the NPV of Amsacta albistriga.

The urticating apparatus in the caterpillar of Lonomia obliqua (Lepidoptera: Saturniidae).

The presence of specialized cells for venom production in the Lonomia obliqua caterpillar has long been a controversial topic. In this study, we identify a cell inside the spine that specializes in the production of toxins. Our histological study showed that this glandular cell was inserted at the subapical region of the spine, in a constricted region like a ring. This cell type was not observed in all spines of the scolus. The constricted region of the spine observed by scanning electron microscopy displayed a circular groove in which the apical portion of the spine fits perfectly; however, some spines in the same scolus lacked this groove. After breaking off the spine at the most apical region, a small drop of orange or green liquid was observed to flow from its tip. These secretions were analysed by MALDI-ToF and found to possess biochemically different compositions. The green secretion demonstrated greater similarity to the haemolymph of the caterpillar than the orange secretion. Based on our findings, the spines with a groove probably contain the venom glands and produce an orange secretion. However, it is also possible that both secretions play an important role in envenoming because all spines in contact with the skin of the accidental victim should break regardless of whether they are present in a groove.