Bacillus thuringiensis Cry1Ac toxin and protoxin do not provoke acute or chronic cytotoxicity on macrophages and leukocytes.

The bioinsecticidal Cry1Ac proteins (protoxin and toxin) are potent immunogens that can activate macrophages by inducing upregulation of costimulatory molecules, pro-inflammatory cytokines, and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, by the oral route, Cry1Ac toxin is mildly allergenic and induces intestinal lymphoid hyperplasia in mice. Given the potential utility of Cry1Ac protoxin as an adjuvant, as well as the human consumption of Cry1Ac toxin in transgenic crops, it is necessary to more deeply evaluate the toxicological potential of these proteins in mammalian immune cells. Here, were used in vitro evaluations in leukocyte and macrophage cell lines to test the potential toxicity of various doses of Cry1Ac proteins, by means of Alamar Blue, MTT, Annexin V, and JC1 assays. Our results indicated that neither Cry1Ac protoxin nor toxin elicited acute toxic effects, after monitoring the cell activity for 4, 8, 10, and 24 h of exposure. By flow cytometry and confocal microscopy analysis, it was observed that neither Cry1Ac toxin nor protoxin generated mitochondrial damage or depolarization or induced apoptosis or necrosis. In conclusion, despite their immunostimulatory effects, it was demonstrated that Cry1Ac proteins did not have cytotoxic effects, even at high concentrations, in primary leukocytes or macrophages or cell lines.

Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc-Gamma-II/and Fc-Gamma-III Receptors Associated with IgG in the Intestinal Epithelium of Mice.

In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ-R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ-RII (CD32) and Fcγ-RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co-localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross-immunoprecipitation results with anti-CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ-RII and Fcγ-RIII and contained IgG associated with Fcγ-RII/RIII. Using anti-CD32 and anti-CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ-RII and Fcγ-RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti-CD16/32, anti-CD16 and anti-CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ-RII and Fcγ-RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice.

Coadministration of protoxin Cry1Ac from Bacillus thuringiensis with metacestode extract confers protective immunity to murine cysticercosis.

The Bacillus thuringiensis Cry1Ac protoxin (pCry1Ac) is a promising mucosal immunogen and adjuvant that induces protective immunity against Naegleria fowleri and malaria infection models. We determined whether pCry1Ac acted as a protective adjuvant against infection with Taenia crassiceps. BALB/C mice were thrice i.p. immunized with (i) pCry1Ac, (ii) metacestode extract, (iii) extract + pCry1Ac or (iv) vehicle, challenged with metacestodes on day 26 and then sacrificed 35 days later. Cysticerci in the peritoneal cavity were counted, while the serum antibody response and cytokines were analysed after immunization and during infection. Only immunization with pCry1Ac plus extract conferred a significant protection (up to 47%). This group presented fluctuating antibody peaks during infection and the highest IgG1 and IgM titres. Immunization with extract alone elicited high IgG1 and the highest IgG2a responses after 25 days of infection, while nonimmunized mice presented a poor, mixed-Th1/Th2 response during infection. Sharp peaks of TNFα and IFN-γ occurred immediately after the first immunization with extract, especially in the presence of pCry1Ac, but not after the challenge, while in the control and pCry1Ac-alone groups, cytokines were only detected after the challenge. The data support the protective-adjuvant effect of co-administration of pCry1Ac in cysticercosis.

Sodium caseinate induces mouse granulopoiesis.

OBJECTIVE AND DESIGN: Sodium caseinate (CasNa) induces differentiation and M-CSF production in mouse band granulocytes in vitro; however, it is not yet known if this molecule can also induce the proliferation and activation of the granulocyte lineage in vivo. In this work we evaluated the induction in vivo of granulopoiesis and the activation of granulocytes in mice treated with CasNa. MATERIAL OR SUBJECTS: BALB/c male mice 8-12 weeks old were used. TREATMENT: The animals were inoculated intraperitoneally with 1 ml of CasNa (10% in PBS p/v) four times (every 48 h). METHODS: Granulocyte proliferation was evaluated by flow cytometry; activation was evaluated by phagocytic indices. The cytokine was measured using an ELISA assay. RESULTS: We show that CasNa increased bone marrow granulopoiesis percentage (38.35 ± 10.88 vs. 64.94 ± 34.14 BrdU+/Gr-1+ cells) and the granulocytes generated presented increased phagocytic indices (0.3 ± 0.1 vs. 0.6 ± 0.11, p < 0.05). We also show that G-CSF (974 ± 411 vs. 3189 ± 350 pg/ml, p < 0.05) and GM-CSF increased in serum, but only G-CSF in bone marrow plasma. CONCLUSIONS: CasNa induces granulopoiesis with functional granulocytes, suggesting that this molecule could be an innate immune system activator.

High glucose concentrations alter the biomineralization process in human osteoblastic cells.

Diabetes mellitus (DM) may alter bone remodeling, as osteopenia and osteoporosis are among the complications. Moreover, DM increases the risk and severity of chronic inflammatory periodontal disease, in which bone resorption occurs. Broad evidence suggests that chronic inflammation can contribute to the development of DM and its complications. Hyperglycemia is a hallmark of DM that may contribute to sustained inflammation by increasing proinflammatory cytokines, which are known to cause insulin resistance, via toll-like receptor (TLR)-4-mediated mechanisms. However, the mechanisms by which bone-related complications develop in DM are still unknown. Studies done on the effect of high glucose concentrations on osteoblast functions are contradictory because some suggest increases (although others suggest reductions) in the biomineralization process. Therefore, we evaluated the effect of high glucose levels on biomineralization and inflammation markers in a human osteoblastic cell line. Cells were treated with either physiological 5.5 mM or increasing concentrations of glucose up to 24 mM, and we determined the following: i) the quantity and quality of calcium-deposit crystals in culture and ii) the expression of the following: a) proteins associated with the process of biomineralization, b) the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), c) cytokines IL1, IL6, IL8, IL10, MCP-1 and TNF alpha, and d) TLR-2, -3, -4 and -9. Our results show that high glucose concentrations (12 mM and particularly 24 mM) alter the biomineralization process in osteoblastic cells and provoke the following: i) a rise in mineralization, ii) an increase in the mRNA expression of RANKL and a decrease of OPG, iii) an increase in the mRNA expression of osteocalcin, bone sialoprotein and the transcription factor Runx2, iv) a diminished quality of the mineral, and v) an increase in the expression of IL1beta, IL6, IL8, MCP-1 and IL10 mRNAs. In addition we found that both high glucose levels and hyperosmotic conditions provoked TLR-2, -3, -4 and -9 overexpression in osteoblastic cells, suggesting that they are susceptible to osmotic stress.

Expression of toll-like receptors 2, 4 and 9 is increased in gingival tissue from patients with type 2 diabetes and chronic periodontitis.

BACKGROUND AND OBJECTIVE: Broad evidence indicates that diabetes both increases the risk and hastens the progression of periodontal disease. Likewise, chronic inflammation or infections seem to provoke insulin resistance and thereby contribute to the development of diabetes and its complications. Innate immune responses, which appear to be altered in individuals with diabetes, are usually mediated by the recognition of pathogens through toll-like receptors (TLRs). The constitutive expression of some TLRs has been reported in healthy human gingival tissue. Interestingly, the expression of TLRs 2 and 4 is increased with the severity of periodontal disease. Considering that the inflammatory reaction is exacerbated in individuals with diabetes and periodontitis, we suspected that the expression of some TLRs might be increased in gingival tissue in these patients. MATERIAL AND METHODS: In this study, we analyzed, by immunofluorescence, the expression of TLRs 2, 3, 4 and 9 in gingival tissues from healthy individuals and from periodontal patients with or without type 2 diabetes. RESULTS: We found that the expression levels of TLRs 2, 3, 4 and 9 were higher in all periodontal patients than in healthy individuals. The expression of some TLRs was increased in subjects with periodontitis and diabetes relative to subjects with periodontitis but without diabetes; this increase in expression was found particularly in TLR2 and TLR9 in the connective tissue and in TLR4 at the epithelial region. CONCLUSION: These data suggest that the expression of these TLRs 2, 3, 4 and 9 in gingival tissue is higher in individuals with diabetes because its inflammatory reaction is exacerbated. Additionally, the expression of these TLRS is positively regulated with the severity of periodontal disease.

Protection against Naegleria fowleri infection in mice immunized with Cry1Ac plus amoebic lysates is dependent on the STAT6 Th2 response.

We previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6-induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6-deficient (STAT6-/-) and wild-type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6-/- mice. Significantly higher titres of Th2-associated IgG1 as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in STAT6-/- mice significantly more IL-12 and IFN-gamma as well as significantly higher titres of Th1-associated IgG2a were detected. Thus, whereas protected STAT6+/+-immunized mice elicited a Th-2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6-/- mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6-signalling pathway is critical for defence against N. fowleri infection.

Striking activation of NALT and nasal passages lymphocytes induced by intranasal immunization with Cry1Ac protoxin.

Cry1Ac protoxin from Bacillus thuringiensis is a potent mucosal immunogen and adjuvant. When delivered intranasally (i.n.) Cry1Ac elicits significant antibody response and is able to improve vaccination against Naegleria fowleri infection, but the functional effects occurring in nasal lymphocytes when this protein is administered alone have not been determined. Here, we investigated the effects of i.n. immunization with Cry1Ac on antibody production, lymphocyte activation and cytokine production in lymphocytes from nasal-associated lymphoid tissue (NALT) and nasal passages (NP). Our results show that i.n. immunization with Cry1Ac induced significant specific IgA and IgG cell responses, especially in NP. Besides, it increased the proportion of lymphocytes expressing the activation markers CD25 and CD69 in both nasal tissues, but differently. CD25 was increased in B cells along with CD4 and CD8 T cells from NALT and NP, while CD69 was increased in B cells from both tissues but only in CD4 T cells from NP. Finally, we found that Cry1Ac augmented especially a Th2 profile of cytokines, as the proportion of T cells that spontaneously produced IL-4, IL-5 and IL-10 was increased and this effect was higher in NP than in NALT. These data contribute to explain the potent immunogenicity of Cry1Ac via i.n. route.

Immunization with Cry1Ac from Bacillus thuringiensis increases intestinal IgG response and induces the expression of FcRn in the intestinal epithelium of adult mice.

We have shown that Cry1Ac protoxin from Bacillus thuringiensis is a potent mucosal and systemic immunogen with adjuvant properties. Interestingly, we have observed that Cry1Ac preferentially induces high specific IgG responses in intestinal fluid when it is intraperitoneally administered to mice; therefore, in the present study, we used this protocol, as a model to address the influence of systemic immunization on the induction of the intestinal IgG response. The data shown indicate that upon intraperitoneal immunization with Cry1Ac, significant intestinal specific IgG cell responses were produced in the lamina propria, accompanied by an increased frequency of intestinal IgG+ lymphocytes and epithelial cells containing IgG. Considering that FcRn is the receptor responsible for the transport of IgG in neonatal intestinal epithelia, but it is developmentally downregulated in the rodent intestine, we analysed whether upon intestinal IgG induction, FcRn mRNA expression was induced in intestinal epithelial cells, of adult mice. Whereas in intestinal epithelia of unimmunized adult mice FcRn mRNA was not detected, in Cry1Ac immunized mice it was expressed, although the level was lower in comparison with that found in neonatal epithelia. Then using flow cytometry and immunofluorescence we confirmed that the expression of the protein FcRn was induced in the intestines of adult immunized mice especially in the large intestine. Finally, we found that Cry1Ac also increased FcRn expression in isolated intestinal epithelial cells stimulated in vitro. The outcomes suggest that the expression of FcRn in intestinal epithelium might be reactivated upon immunization, and possibly facilitate IgG transport.

Intranasal immunization with Naegleria fowleri lysates and Cry1Ac induces metaplasia in the olfactory epithelium and increases IgA secretion.

According to previous reports, intranasal administration of the Cry1Ac protein alone or with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice, apparently by eliciting IgA responses in the nasal mucosa. In the current study, we performed an immunohistochemical analysis of IgA in the nasal mucosa of mice immunized intranasally with Cry1Ac, and amoebic lysates or a combination of both. The animals were sacrificed 24 h after the last immunization or after an intranasal lethal challenge with N. fowleri. Our results indicate that all of the intranasal immunizations provoked an increase in areas with metaplasia in the olfactory epithelium, allowing for secretion of IgA. As a result, IgA antibodies were found interacting with trophozoites in the nasal lumen, and there was a marked increase of IgA in the metaplasic epithelium. On the other hand in nonimmunized mice trophozoites were observed invading the nasal mucosa, which was not the case for immunized mice. Our results suggest that intranasal immunization provokes cellular changes in the olfactory epithelium, leading to greater protection against N. fowleri that is probably caused by an increased secretion of IgA. The increased IgA response induced in the nasal mucosa by immunization probably impedes both amoebic adhesion and subsequent invasion of the parasite to the nasal epithelium.

Carrier potential properties of Bacillus thuringiensis Cry1A toxins for a diphtheria toxin epitope.

The N-terminal half or toxic fragment of Bacillus thuringiensis Cry proteins is comprised of three structural domains. In a previous paper, we showed that this region plays an important role in the immunogenicity of the B. thuringiensis Cry proteins. Due to this ability and along with their stability it is worthy of investigating whether this region has carrier potential. To approach this, an eight amino acid hydrophobic motif in alpha-helix 7 of wild-type (WT) Cry1A toxins was exchanged for a diphtheria toxin epitope (DTB). The resultant recombinant toxins were tested for their ability to induce specific anti-Cry and anti-diphtheria toxin antibodies in mice after intraperitoneal and nasal immunization. We found that recombinant Cry1A toxins retained their ability to induce serum and mucosal anti-Cry Ab as well as IgG subclasses, although with a varied magnitude. By the systemic route, the effect of the amino acid substitution in the ratio of the IgG1/IgG2a Ab, leading in some sites toward IgG1 or IgG2a is more evident. Interestingly, mice produced specific anti-DTB IgG, and IgA after intranasal immunization. Together, our results support and show the immunogenic properties of the WT Cry1A toxins as well as its carrier potential for a DTB.

Analysis of the cellular immune response induced by Bacillus thuringiensis Cry1A toxins in mice: effect of the hydrophobic motif from diphtheria toxin.

Insecticidal Cry1A toxins from Bacillus thuringiensis elicit strong humoral immune response in mice. Previously, we showed that an eight hydrophobic motif amino acid substitution in Domain I did not affect the antibody inducing capacity of the Cry1A toxins, on the contrary, it was enhanced after intranasal immunization. In addition, Cry1A mutants (carrying a substitution of a motif from fragment B of diphtheria toxin into the structurally similar hydrophobic alpha-helix 7 motif of Cry1A toxins) were able to modulate the ratio of IgG subclasses, IgG1/IgG2a. However, the capacity of these toxins to induce cellular immune response has not been studied. Thus, in this work, we investigated the cytokine profile induced after in vitro stimulation with the toxins, in spleen cell cultures from unprimed mice, and intranasally primed mice, with either wild-type Cry1Aa or with mutant toxin Cry1Aa8. Spleen cells from unprimed mice stimulated with Cry1Aa produced very low levels of Th1 (IFN-gamma, IL-12p70) and Th2 type cytokines (IL-10, IL-4), whereas immunization with Cry1Aa8 toxin led to higher production of these cytokines. Restimulation of spleen cells from primed mice with the Cry1Aa induced the production of significant levels of IL-12p70 whereas with Cry1Aa8, IFN-gamma production was stimulated. Interestingly, we found that the capacity of Cry1A toxins to induce cytokine production by lymphocytes was inhibited by N-acetylgalactosamine. Altogether these data demonstrate the immunogenic properties of Cry1A toxins and show that amino acid substitution in Domain I principally affects its ability to induce Th1 cytokines in lymphocytes.

Intranasal Cry1Ac protoxin is an effective mucosal and systemic carrier and adjuvant of Streptococcus pneumoniae polysaccharides in mice.

Streptococcus pneumoniae is a major respiratory pathogen in infants, children and the elderly. Available parenteral anti-pneumococcal vaccines based on type-specific capsular polysaccharides (CPSs) are useful in adults but do not elicit protective immunity in infants and young children. To enhance their immunogenicity, pneumococcal CPSs conjugated to proteins are being developed. Mucosal vaccines may induce mucosal and systemic immune responses, but their development has been hampered by the lack of effective, inexpensive innocuous mucosal adjuvants or immunogenic vaccine carriers. We have demonstrated that the recombinant Cry1Ac protoxin from Bacillus thuringiensis is highly immunogenic and has mucosal and systemic adjuvant effects on proteins coadministered in mice. In this work, we evaluated Cry1Ac as a carrier and adjuvant of S. pneumoniae CPS for the induction of mucosal and systemic antibody responses after intranasal and intraperitoneal immunization in mice. Our results demonstrate that intranasal application of pneumococcal polysaccharides either coadministered or conjugated with Cry1Ac induces higher systemic and mucosal specific antibody responses than those elicited by pneumococcal polysaccharides alone. Adjuvant effects of Cry1Ac on polysaccharides may be appropriate for vaccine design.

Differences between the large and small intestine in the immunodominant amoebic proteins recognized by IgG and IgA antibodies in BALB/c mice.

We have previously reported that there are differences in the number of predominant amoebic antigens recognized by serum and small intestinal antibodies induced after local and systemic immunization with glutarldehyde-fixed Entamoeba histolytica trophozoites (GFT) in BALB/c mice, by an immunoblot analysis. Moreover, by enzyme-linked immunosorbent assay (ELISA) analysis, we found differences in the antiamoebic antibody isotype patterns elicited at the large and small intestines. To further characterize the antiamoebic immune response induced in BALB/c mice, after local (oral and rectal) and systemic (intraperitoneal and intramuscular) immunization with GFT, we performed an immunoblot analysis of the amoebic proteins predominantly recognized by immunoglobulins (Ig)G, IgA and IgM in the serum and in the small and large intestines. The present work shows differences between the large and small intestine in the IgG- and IgA-antibody recognition pattern of amoebic proteins, thus confirming and extending our previous findings supporting the compartmentalization of the intestinal immune response. Furthermore, our reported observation that there are differences in the amoebic proteins predominantly recognized by antibodies of different isotypes was extended to the intestines, as some proteins with relative molecular weights of 24-25, 66, 140 kDa are strongly recognized by IgG but not by other antibody isotypes.

Intranasal, rectal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis induces compartmentalized serum, intestinal, vaginal and pulmonary immune responses in Balb/c mice.

Recently we discovered that the Cry1Ac protoxin of Bacillus thuringiensis administered to Balb/c mice intraperitoneally (i.p.) or intragastrically is a systemic and intestinal immunogen as potent as cholera toxin. To further characterize the mucosal immunogenicity of Cry1Ac we additionally tried the intranasal (i.n.) and rectal routes and used enzyme-linked immunoassays to determine anti-Cry1Ac antibody responses in the serum as well as in vaginal and tracheobronchial washes and in the fluids of the large and the small intestine. Immunization by the i.p., i.n. and rectal routes induced IgM, IgG and IgA antibodies in all the mucosal surfaces analyzed, but the magnitude and predominant isotype of each response depended on the route used and the mucosal site analyzed. These data extend our findings on the striking mucosal immunogencity of Cry1Ac and provide additional evidence on the compartmentalization of the mucosal immune system.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice.

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins