Ronaldo Z. Mendonça

Type I IFNs Stimulate Nitric Oxide Production and Resistance to Trypanosoma cruzi Infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-αβ or anti-TNF-α Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-α (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-αβ Ab reduced NO production. IFN-γ or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-αβR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-αβ, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.

An insight into insect cell metabolism through selective nutrient manipulation

Nutritional information of insect cell cultures, required for designing strategies to increase protein productivity, is still limited. In this work, nutrient feeding or removal was used to determine the role of selected nutrients and their interactions in insect cell metabolism before viral infection. Glucose, glutamine, tyrosine, and methionine were consumed at the highest rates in batch and fed-batch cultures, and thus were selected to be manipulated. Glucose and glutamine could be replaced by other carbon sources or amino acids, respectively. However, growth was reduced when such alternative sources were utilized, revealing the distinctive role of glucose and glutamine. Glucose and glutamine consumption rates were regulated by their own concentration, although glutamine concentration did not affect glucose consumption rate or vice versa. Excessive glucose or glutamine supply caused energy and carbon wastes, as observed by reduced cell yields and accumulation of by-products, such as alanine. Nevertheless, growth inhibition was attenuated by simultaneous excess of both nutrients, probably because cells had both carbon and ammonia available to deal with toxic by-products in the form of alanine. Other amino acids, such as tyrosine and methionine, were also relevant for maintaining prolonged viability. The importance of an adequate nutrient supply was demonstrated; particularly, the need to maintain glucose and glutamine concentrations just above their critical values.

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding.

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.

Anthelmintic Activity of the Natural Compound (+)-Limonene Epoxide against Schistosoma mansoni

Blood fluke of the genus Schistosoma are the etiological agents of human schistosomiasis, an important neglected tropical disease that afflicts over 200 million people worldwide. The treatment for this disease relies heavily on a single drug, praziquantel. Recent reports of praziquantel resistance raise concerns about future control of the disease and show the importance of developing new antischistosomal drugs. Currently, natural products have been a good source for drug development. (+)-Limonene epoxide is a mixture of cis and trans isomers found in many plants. Here, we report the in vitro effect of this natural compound on the survival time of Schistosoma mansoni adult worms. In addition, we examined alterations on the tegumental surface of adult schistosomes by means of confocal laser scanning microscopy. The effects of (+)-limonene epoxide at 25 µg/mL on S. mansoni adult worms were similar to those of the positive control (praziquantel), with reduction in motility and death of all worms after 120 h. Confocal laser scanning microscopy revealed that (+)-limonene epoxide-mediated worm killing was associated with tegumental destruction. Our results, along with the low toxicity of the (+)-limonene epoxide, suggest that this natural compound might be promising for the development of new schistosomicidal agents.

Development and Antibacterial Activity of Cashew Gum-Based Silver Nanoparticles

The present study describes the development of a green synthesis of silver nanoparticles reduced and stabilized by exuded gum from Anacardium occidentale L. and evaluates in vitro their antibacterial and cytotoxic activities. Characterization of cashew gum-based silver nanoparticles (AgNPs) was carried out based on UV–Vis spectroscopy, transmission electron microscopy and dynamic light scattering analysis which revealed that the synthesized silver nanoparticles were spherical in shape, measuring about 4 nm in size with a uniform dispersal. AgNPs presented antibacterial activity, especially against Gram-negative bacteria, in concentrations where no significant cytotoxicity was observed.

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.

Crotalus durissus terrificus Venom Interferes WithMorphological, Functional, and Biochemical Changes in Murine Macrophage

Crotalus durissus terrificus venom (Cdt) is toxic for a variety of eukaryotic cells, especially at high concentrations. However its effects on host immune cells are not well known. The purpose of this study was to determine the effect of Cdt on functional status and the mediators production in peritoneal macrophages. The effects of Cdt were analyzed in vitro and were detected using functional status of macrophages as determined by the H(2)O(2) release, spreading percentage, phagocytic index, vacuole formation, and mediators production. Several functional bioassays were employed: cytotoxicity was determined by taking the lyses percentage and the presence of hydrogen peroxide (H(2)O(2)) in macrophages, using the horseradish peroxidase-dependent oxidation of phenol red and nitric oxide (NO) in the supernatants of macrophages by the Griess reaction. The tumor necrosis factor (TNF) activity was detected by measuring its cytotoxic activity on L929 cells, and the production the level of other cytokines was assayed using enzyme-linked immunosorbent assay. In vitro studies revealed that Cdt produced (a) a discrete increase in the release of H(2)O(2) and vacuole formation; (b) a decrease in spreading percentage and in the phagocytic index; and (c) an increment in the mediators production. More pronounced increments of IL-6 and TNF were observed after 24 and 48 hours, respectively. Maximum levels of IFN-gamma and NO were observed after 96 hours. Interestingly, levels of all mediators presented a discreet decrease, as the amount of Cdt was increased. In contrast, the IL-10 levels observed for all doses studied here did not alter. The IL-6/IL-10 ratio may possibly reflect the balance of pro- and anti-inflammatory cytokines in macrophages, which may be manifested in the inflammatory status during the envenoming processes. Taken together, these data indicate that Cdt have a differential effect on macrophage activation and that this venom is a potent inhibitor of anti-inflammatory response.Crotalus durissus terrificus venom (Cdt) is toxic for a variety of eukaryotic cells, especially at high concentrations. However its effects on host immune cells are not well known. The purpose of this study was to determine the effect of Cdt on functional status and the mediators production in peritoneal macrophages. The effects of Cdt were analyzed in vitro and were detected using functional status of macrophages as determined by the H2O2 release, spreading percentage, phagocytic index, vacuole formation, and mediators production. Several functional bioassays were employed: cytotoxicity was determined by taking the lyses percentage and the presence of hydrogen peroxide (H2O2) in macrophages, using the horseradish peroxidase-dependent oxidation of phenol red and nitric oxide (NO) in the supernatants of macrophages by the Griess reaction. The tumor necrosis factor (TNF) activity was detected by measuring its cytotoxic activity on L929 cells, and the production the level of other cytokines was assayed using enzyme-linked immunosorbent assay. In vitro studies revealed that Cdt produced (a) a discrete increase in the release of H2O2 and vacuole formation; (b) a decrease in spreading percentage and in the phagocytic index; and (c) an increment in the mediators production. More pronounced increments of IL-6 and TNF were observed after 24 and 48 hours, respectively. Maximum levels of IFN-γ and NO were observed after 96 hours. Interestingly, levels of all mediators presented a discreet decrease, as the amount of Cdt was increased. In contrast, the IL-10 levels observed for all doses studied here did not alter. The IL-6/IL-10 ratio may possibly reflect the balance of pro- and anti-inflammatory cytokines in macrophages, which may be manifested in the inflammatory status during the envenoming processes. Taken together, these data indicate that Cdt have a differential effect on macrophage activation and that this venom is a potent inhibitor of anti-inflammatory response.

Inhibitory potential of Crotalus durissus terrificus venom on measles virus growth

This paper presents the antiviral activity found in a snake with Crotalus durissus terrificus venom (Cdt), studied by use of microplate inhibition assay, using measles virus (MV). Cdt at concentrations below 100 microg/ml showed no cytotoxicity for Vero cells. This study shows the optimal conditions for cell treatment and infection. Two factors that affect virus binding and infection efficiency were studied: the use of an adsorption step, where infection volume was varied; and the concentration of fetal bovine serum (FBS). The adsorption step, with or without FBS, increased the bound virus percentage, whereas it increased bound virus at equilibrium only in FBS-free until 2.5% FBS. In contrast, the addition of 10% FBS decreased the bound virus percentage. The inhibition of MV replication in Vero cells was observed when Cdt was added either before or during cell infection with virus. Its inhibitory concentration against MV replication was 0.1 until 100 microg/ml, respectively. The anti-MV effect of the Cdt was gradually decreased when it was added before or during infection, and little inhibition was observed when Cdt was added 1 h after infection, suggesting that the MV infection was inhibited at the time of the initial events such as at the moment of adsorption and penetration of the viral cycle. In conclusion, Cdt contains anti-MV effects that may be of potential clinical interest.