Ana Lilia García-Hernández

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.

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.

A Plant-Derived Multi-HIV Antigen Induces Broad Immune Responses in Orally Immunized Mice

Abstract Multi-HIV, a multiepitopic protein derived from both gp120 and gp41 envelope proteins of the human immunodeficiency virus (HIV), has been proposed as a vaccine prototype capable of inducing broad immune responses, as it carries various B and T cell epitopes from several HIV strains. In this study, the immunogenic properties of a Multi-HIV expressed in tobacco chloroplasts are evaluated in test mice. BALB/c mice orally immunized with tobacco-derived Multi-HIV have elicited antibody responses, including both the V3 loop of gp120 and the ELDKWA epitope of gp41. Based on splenocyte proliferation assays, stimulation with epitopes of the C4, V3 domain of gp120, and the ELDKWA domain of gp41 elicits positive cellular responses. Furthermore, specific interferon gamma production is observed in both CD4+ and CD8+ T cells stimulated with HIV peptides. These results demonstrate that plant-derived Multi-HIV induces T helper-specific responses. Altogether, these findings illustrate the immunogenic potential of plant-derived Multi-HIV in an oral immunization scheme. The potential of this low-cost immunization approach and its implications on HIV/AIDS vaccine development are discussed.

Immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic HIV protein.

Elicitation of broad humoral immune responses is a critical factor in the development of effective HIV vaccines. In an effort to develop low-cost candidate vaccines based on multiepitopic recombinant proteins, this study has been undertaken to assess and characterize the immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic protein. This protein consists of V3 loops corresponding to five different HIV isolates, including MN, IIIB, RF, CC, and RU. In this study, both Escherichia coli and lettuce-derived C4(V3)6 have elicited local and systemic immune responses when orally administered to BALB/c mice. More importantly, lettuce-derived C4(V3)6 has shown a higher immunogenic potential than that of E. coli-derived C4(V3)6. Moreover, when reactivity of sera from mice immunized with C4(V3)6 are compared with those elicited by a chimeric protein carrying a single V3 sequence, broader responses have been observed. The lettuce-derived C4(V3)6 has elicited antibodies with positive reactivity against V3 loops from isolates MN, RF, and CC. In addition, splenocyte proliferation assays indicate that significant T-helper responses are induced by the C4(V3)6 immunogen. Taken together, these findings account for the observed elicitation of broader humoral responses by the C4(V3)6 multiepitopic protein. Moreover, they provide further validation for the production of multiepitopic vaccines in plant cells as this serves not only as a low-cost expression system, but also as an effective delivery vehicle for orally administered immunogens.

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.

The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.

Production of an antigenic C4(V3)6 multiepitopic HIV protein in bacterial and plant systems

A C4(V3)6 multiepitopic protein was designed in an effort to pursue broad immunization against the human immunodeficiency virus (HIV). This C4(V3)6 chimeric protein is based on sequences of gp120, including epitopes from the fourth conserved domain (C4) and six tandem repeats of the third variable domain (V3), which represent different HIV isolates. The histidine-tagged C4(V3)6 was subsequently over-expressed in a recombinant Escherichia coli strain, and purified by immobilized metal ion affinity chromatography. Expression of the C4(V3)6 in both tobacco and lettuce plants was also achieved with no toxic effects on plant growth as transgenic plants were phenotypically normal. Moreover, the functional C4(V3)6 protein showed HIV antigenic determinants. The implications of these findings on the development of a new low-cost HIV vaccine are discussed.

Mucosal Immunology and Oral Vaccination

Mucosal surfaces are exposed to a wide variety of antigens and represent the major access route for pathogens. The immune system at the distinct mucosal compartments is highly specialized and regulated as it has to distinguish between inoffensive antigens and potential pathogens. Mucosal vaccines offer important advantages over the widely used parenteral ones since they constitute the best strategy to stimulate protective immune responses at the mucosal-invading pathogen routes, which have been evidenced with the oral polio vaccine or other replicating vaccines. However, the development of mucosal vaccines, particularly subunit vaccines, has showed limited success because eliciting effective immune responses represents a significant challenge because the vaccine has to overcome the complex mucosal environment and also deal with the immune regulation mechanisms that maintain the homeostasis. Fortunately, advances in the knowledge of the organization and function of the mucosal immune system, which is basically divided into inductive and effector sites, have provided important clues for the design of mucosal vaccines. Particularly, the oral immunization route is still considered the safest route, and plant-based vaccines may represent a promising strategy to overcome obstacles related to the administration of antigens by this route since plant biomass possesses characteristics that influence the efficiency of delivery at the mucosal inductive sites as well as compounds that may serve as adjuvants to mount effective mucosal immune responses.

Study of the allergenic potential of Bacillus thuringiensis Cry1Ac toxin following intra-gastric administration in a murine model of food-allergy

&NA; Cry1Ac toxin, from Bacillus thuringiensis, is widely used as a biopesticide and expressed in genetically modified (GM) plants used for human and animal consumption. Since Cry1Ac is also immunogenic and able to activate macrophages, it is crucial to thoroughly evaluate the immunological effects elicited after intra‐gastric administration. The allergenic potential of purified Cry1Ac was assessed and compared with that induced in a murine model of food‐allergy to ovalbumin (OVA), in which animals are sensitized with the adjuvant Cholera toxin (CT). Mice were weekly intragastrically administered with: i) vehicle phosphate‐buffered saline (PBS), ii) OVA, iii) OVA plus CT iv) Cry1Ac or v) OVA plus Cry1Ac. Seven weeks after, mice were intragastrically challenged and allergic reactions along with diverse allergy related immunological parameters were evaluated at systemic and intestinal level. The groups immunized with, Cry1Ac, OVA/Cry1Ac or OVA/CT developed moderate allergic reactions, induced significant IgE response and increased frequencies of intestinal granulocytes, IgE+ eosinophils and IgE+ lymphocytes. These same groups also showed colonic lymphoid hyperplasia, notably in humans, this has been associated with food allergy and intestinal inflammation. Although the adjuvant and allergenic potential of CT were higher than the effects of Cry1Ac, the results show that applied intra‐gastrically at 50 &mgr;g doses, Cry1Ac is immunogenic, moderately allergenic and able to provoke intestinal lymphoid hyperplasia. Moreover, Cry1Ac is also able to induce anaphylaxis, since when mice were intragastrically sensitized with increasing doses of Cry1Ac, with every dose tested, a significant drop in rectal temperature was recorded after intravenous challenge. Graphical abstract: Figure. No caption available. HighlightsMildly allergic reactions elicited by the intra‐gastric administration of Cry1Ac.Cry1Ac induced increase of IgE response and proportion of B220+ IgE+ lymphocytes.Cry1Ac induced increment of eosinophils and granulocytes in the intestine.Lymphoid intestinal hyperplasia provoked by intra‐gastric Cry1Ac.Cry1Ac toxin induced anaphylaxis after intravenous challenge.