Differential capability of Bacillus thuringiensis Cry1Ac protoxin and toxin to induce in vivo activation of dendritic cells and B lymphocytes.

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant--Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation.

In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt) Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o.) or with a single intraperitoneal (i.p.) injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed). This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.

Evaluation of cytotoxic and antimicrobial effects of two Bt Cry proteins on a GMO safety perspective.

Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability (IC50 > 1,000 µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis (IC50 > 1,000 µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested (MIC > 1,000 µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications.

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton.

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.

Pretreatment with Cry1Ac protoxin modulates the immune response, and increases the survival of Plasmodium-infected CBA/Ca mice.

Malaria is a major global health problem that kills 1-2 million people each year. Despite exhaustive research, naturally acquired immunity is poorly understood. Cry1A proteins are potent immunogens with adjuvant properties and are able to induce strong cellular and humoral responses. In fact, it has been shown that administration of Cry1Ac protoxin alone or with amoebic lysates induces protection against the lethal infection caused by the protozoa Naegleria fowleri. In this work, we studied whether Cry1Ac is able to activate the innate immune response to induce protection against Plasmodium berghei ANKA (lethal) and P. chabaudi AS (nonlethal) parasites in CBA/Ca mice. Treatment with Cry1Ac induced protection against both Plasmodium species in terms of reduced parasitaemia, longer survival time, modulation of pro- and anti-inflammatory cytokines, and increased levels of specific antibodies against Plasmodium. Understanding how to boost innate immunity to Plasmodium infection should lead to immunologically based intervention strategies.

Adjuvant effects of crystal proteins from a Mexican strain of Bacillus thuringiensis on the mouse humoral response.

In this study, we determined the adjuvant effects of the crystal (Cry) proteins, p130, p98, and p64-62, on the immune response of mice to both sheep red blood cells (SRBC) and ovalbumin (OVA). The administration of p130, p98, and p64-62 Cry proteins to Balb/c mice induced a significant (p<0.01) increase in the production of anti-SRBC antibody-secreting cells (ASC). The p64-62 Cry proteins demonstrated the best ability to induce the production of IgA and IgG antibodies to SRBC (p<0.05), and IgM, IgA, and IgG antibodies to OVA (p<0.05). Additionally, Cry proteins did not produce any side effects associated with their administration to Balb/c mice. We suggest the potential use of the p64-62 Cry proteins as adjuvants for the administration of heterologous antigens.

[Adjuvant effect of the Cry1Ac protoxin on cellular immunity when is coadministered with a good mucosal pathogen]. / Efecto coadyuvante de la protoxina Cry1Ac en la inmunidad celular al administrarla con un buen agente patógeno de mucosas.

BACKGROUND: The major part of pathogens use the mucosae to penetrate into the organisms, the idea to blockade the invasion, replication, colonization and multiplication of them results attractive not only to vaccines design, but immunopharmacs design, too. The protoxin Cry1Ac from Bacillus thuringiensis is a recent known mucosal adjuvant. OBJECTIVE: To determine the adjuvant effect of protoxin CrylAc from Bacillus thuringiensis on cellular response. MATERIAL AND METHODS: Mononuclear cells from human peripheral blood were isolated in Ficoll-Hypaque gradients (d=1077), those cells CD3-CD14+ were obtained with the help of FACSVantage system, and then were stimulated with a lisate of S. pyogenes alone or combined with different concentrations of CrylAc. The proliferation of monocytes was determined in experiments of timidina-3H incorporation. RESULTS: The proliferative response of monocytes was major in those stimulated with S. pyogenes lisate and CrylAc than the observed when monocytes were stimulated with lisate alone. The major response was obtained with lisates of S. pyogenes plus 12.5 microg/mL of CrylAc (p<0.05). CONCLUSION: CrylAc have an important adjuvant effect on the cellular immune response. These findings are important to treat infections and for vaccines design.

Long-lasting effects of a Bacillus thuringiensis serovar israelensis experimental tablet formulation for Aedes aegypti (Diptera: Culicidae) control.

Dengue is a growing public health problem in many tropical and subtropical countries worldwide. At present, the only method of controlling or preventing the disease is to eliminate its vector, Aedes aegypti (L.) (Diptera: Culicidae). In the current study, an experimental larvicide tablet formulation XL-47 based on Bacillus thuringiensis serovar israelensis (Bti) and containing 4.8% of technical powder was developed. This formulation was evaluated against Ae. aegypti in three different sets of experiments, under field-simulated conditions: two experiments were indoors and under partial sunlight exposure and one experiment was outdoors with sunlight exposure. Larvae were added throughout the experiment two times per week, and the residual larvicidal activity was recorded daily. Pupal formation was reduced in the containers with Bti by > 80% in relation to the containers without treatment for 12 wk; to our knowledge, this is the longest period of control reported for a Bti tablet formulation outdoors under sunlight exposure. Moreover, samples from the top, middle, and bottom of the water column were collected to perform bacterial plate counts and toxicity assays. The Bti population and the active ingredient of the tablet formulation remained mainly at the bottom of the containers and mosquito larvae reached the formulation by diving and shredding the tablet's material. In conclusion, the experimental tablet formulation XL-47 showed an inhibition of pupal formation that lasted for long periods under sunlight exposure.