Juan Manuel Alcocer-González

A Novel Mucosal Vaccine Based on Live Lactococci Expressing E7 Antigen and IL-12 Induces Systemic and Mucosal Immune Responses and Protects Mice against Human Papillomavirus Type 16-Induced Tumors

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When challenged with lethal levels of tumor cell line TC-1 expressing E7, immunized mice showed full prevention of TC-1-induced tumors, even after a second challenge, suggesting that this prophylactic immunization can provide long-lasting immunity. Therapeutic immunization with L. lactis recombinant strains, i.e., 7 days after TC-1 injection, induced regression of palpable tumors in treated mice. The antitumor effects of vaccination occurred through a CTL response, which is CD4+ and CD8+ dependent. Furthermore, immunized mice developed an E7-specific mucosal immune response. These preclinical results suggest the feasibility of the low-cost mucosal vaccination and/or immunotherapy strategies against HPV-related cervical cancer in humans.

Antitumor activity of colloidal silver on MCF-7 human breast cancer cells

BackgroundColloidal silver has been used as an antimicrobial and disinfectant agent. However, there is scarce information on its antitumor potential. The aim of this study was to determine if colloidal silver had cytotoxic effects on MCF-7 breast cancer cells and its mechanism of cell death.MethodsMCF-7 breast cancer cells were treated with colloidal silver (ranged from 1.75 to 17.5 ng/mL) for 5 h at 37°C and 5% CO2 atmosphere. Cell Viability was evaluated by trypan blue exclusion method and the mechanism of cell death through detection of mono-oligonucleosomes using an ELISA kit and TUNEL assay. The production of NO, LDH, and Gpx, SOD, CAT, and Total antioxidant activities were evaluated by colorimetric assays.ResultsColloidal silver had dose-dependent cytotoxic effect in MCF-7 breast cancer cells through induction of apoptosis, shown an LD50 (3.5 ng/mL) and LD100 (14 ng/mL) (*P < 0.05), significantly decreased LDH (*P < 0.05) and significantly increased SOD (*P < 0.05) activities. However, the NO production, and Gpx, CAT, and Total antioxidant activities were not affected in MCF-7 breast cancer cells. PBMC were not altered by colloidal silver.ConclusionsThe present results showed that colloidal silver might be a potential alternative agent for human breast cancer therapy.

Cell-surface display of E7 antigen from human papillomavirus type-16 in Lactococcus lactis and in Lactobacillus plantarum using a new cell-wall anchor from lactobacilli.

The human papillomavirus type-16 (HPV-16) E7 protein is considered a major viral oncoprotein involved in cervical cancer (CxCa) and a potential candidate for the development of a vaccine against this neoplasia. Here, two lactic acid bacteria (the model one Lactococcus lactis and a probiotic one Lactobacillus plantarum) were engineered to deliver an E7 mutant protein (E7mm), which has a reduced transforming activity and consequently, could fit better to therapeutic use in humans than the native form of E7. An efficient cell-surface display of E7mm was obtained in L. lactis using an expression cassette encoding a precursor composed of (i) the signal peptide and the first 15 amino acids of the mature part of the lactococcal Usp45 protein; (ii) E7mm and (iii) the cell-wall anchor of the Streptococcus pyogenes M6 protein (CWAM6). This hybrid precursor was produced but not cell-wall anchored in Lb. plantarum. We thus replaced CWAM6 by the cell-wall anchor of a Lb. plantarum protein which allows an efficient cell-wall anchoring of E7mm in this bacterium. The E7mm production and cell-surface display in both L. lactis and a probiotic bacterium, Lb. plantarum, represent one more step towards the development of a safe and effective treatment against CxCa.

Detection of genes encoding antimicrobial peptides in Mexican strains of Trichoplusia ni (Hübner) exposed to Bacillus thuringiensis

The systemic immune response of Trichoplusia ni after Bacillus thuringiensis (Bt) exposure was evaluated by comparing the expression of genes encoding antimicrobial peptides (AMPs) in Bt-susceptible and -resistant T. ni strains that were either exposed or not to XenTari (Bt-XT). AMP genes were detected by RT-PCR using primers for attacin, gloverin, lebocin, lysozyme, and peptidoglycan recognition peptide (PGRP). In general, AMP genes were detected more frequently in Mexican field strains previously exposed to Bt (SALX and GTOX) than in a Mexican laboratory strain (NL), but expression was similar to the AMP expression in USA laboratory strains (US and USX). Among the AMPs, transcripts for lebocin were the least detected (11.7%) and those for lysozyme were the most detected (84.8%) in all samples. Lebocin was detected only in 2nd instar and pupa. All untreated controls expressed attacin. Attacin and gloverin were not detected in any midgut sample, and their highest detection was in pupa. Lysozyme was rarely detected in 2nd instar larvae from any strain or treatment but was detected in almost all midgut and hemolymph samples. Overall, AMPs were found more in T. ni strains previously exposed to Bt-XT, especially lebocin and globerin (1.8-fold increase) and PGRP (3.8-fold increase). The data suggest that the expression of AMPs in T. ni correlates to previous Bt exposure.

Comparative Evaluation of Phenoloxidase Activity in Different Larval Stages of Four Lepidopteran Pests After Exposure to Bacillus thuringiensis

Abstract Microbial entomopathogen—based bioinsecticides are recognized as alternatives to synthetic pesticides. Insects defend themselves against microbial pathogens by innate mechanisms, including increased phenoloxidase (PO) activity, but its relationship with microbial bioinsecticides efficacy is little known. This study evaluated the differences in PO activity at different developmental stages of the tobacco budworm Heliothis virescens Fabricius (Lepidoptera: Noctuidae), Indian meal moth Plodia interpunctella (Hübner) (Pyralidae), beet armyworm Spodoptera exigua (Hübner) (Noctuidae), and cabbage looper Trichoplusia ni (Hübner) (Noctuidae). Additionally, 2nd- and 4th-instars were exposed to the LC50 value of the commercial Bacillus thuringiensis (Bt) spray, Biobit®. The percentage of insecticidal activity (IA%) on 2nd-instar Biobit—exposed larvae was approximately the predicted 50 % mortality for all species except S. exigua. With all 4th instar Biobit—exposed larvae, mortality was not significantly different from that of unexposed larvae. Unexposed insects had a significantly higher PO activity in pre—pupae and pupae than early—instar larvae and adults, whereas PO activity was higher in adult females than in males. Correlation analysis between IA% and PO activity revealed significant r—values (p < 0.01) in 2nd instar H. virescens (r = 0.979) and P. interpunctella (r = 0.930). Second instar Biobit—exposed P. interpunctella had 10 times more PO activity than unexposed larvae. Similarly, the amount of total protein was lower in 4th instar Biobit—exposed H. virescens and higher in S. exigua. Therefore, the results indicated a relationship between Biobit susceptibility and PO activity in some cases. This information may be useful if the Biobit application period is timed for a developmental stage with low PO activity. However, more studies are needed to determine the correlation of each insect with a particular bioinsecticide.

CD3ζ Expression and T Cell Proliferation are Inhibited by TGF-β1 and IL-10 in Cervical Cancer Patients

IntroductionCervical cancer development from a squamous intraepithelial lesion is thought to be favored by an impaired T cell immunity. We evaluated parameters of T cell alterations such as proliferation, cytokine, and CD3ζ expression in peripheral blood and tumor-infiltrating T lymphocytes from women with squamous intraepithelial lesions (SIL) or cervical cancer (CC).Results and DiscussionT cell proliferation and cytokine messenger RNA (mRNA) expression were similar in women with SIL and healthy donors, whereas low T cell proliferation and lower mRNA expression of IL-2, IL-10 and IFN-γ were observed in women with CC. Moreover, infiltrating cells showed marginal responses. We also found that CD3ζ mRNA expression, whose protein is required for T cell activation, correlated with a decreased proliferation in advanced stages of the disease.ConclusionExperiments with T cells from healthy donors in the presence TGF-β1 or IL-10 suggest that these cytokines have a relevant role in T cell responses during CC progression.

Effect of 60 Hz electromagnetic fields on the activity of hsp70 promoter: an in vivo study

Exposure to EMFs (electromagnetic fields) results in a number of important biological changes, including modification of genetic expression. We have investigated the effect of 60 Hz sinusoidal EMFs at a magnetic flux density of 80 μT on the expression of the luciferase gene contained in a plasmid labelled as pEMF (EMF plasmid). This gene construct contains the specific sequences for the induction of hsp70 (heat-shock protein 70) expression by EMFs, as well as the reporter for the luciferase gene. The pEMF vector was electrotransferred into quadriceps muscles of BALB/c mice that were later exposed to EMFs. Increased luciferase expression was observed in mice exposed to EMFs 2 h daily for 7 days compared with controls (P<0.05). These data along with other reports in the literature suggest that EMFs can have far-reaching effects on the genome.

Entamoeba histolytica and Trichomonas vaginalis: trophozoite growth inhibition by metronidazole electro-transferred water.

The influence of low-frequency electromagnetic (LF-EM) waves on microorganisms has been a subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting procedure known as biophysical-information-therapy or bioresonance therapy (BRT) which in principle is based on LF-EM stimulation, has emerged. BRT was discovered in the late 1980s but it is still poorly studied. This paper demonstrates that by transferring metronidazole information to water samples by an electronic amplifier (BRT device), the growth of axenically cultured trophozoites of Entamoeba histolytica and Trichomonasvaginalis is significantly inhibited, compared with those cultures treated with non and sham electro-transferred water samples. A positive control of metronidazole, a well-known cytotoxic drug against parasites, was used as a reference.

HPV 16 E2 Protein Induces Apoptosis in Human and Murine HPV 16 Transformed Epithelial Cells and Has Antitumoral Effects in vivo

Objective: Our aims were to examine the ability of the human papillomaviruse (HPV) 16 E2 protein to induce apoptosis in a murine HPV-transformed cell line, and to evaluate its antitumor properties on HPV-associated tumors in vivo in immunocompetent mice. Methods: HPV-transformed murine BMK-16/myc cells and human SiHa cells were transfected with the HPV 16 E2 gene to examine the effects of the E2 protein on cell growth and on the E6 and E7 oncogenes as well as DNA fragmentation and activation of the extrinsic pathway of apoptosis. Finally, to test the antitumor effect of the E2 protein on an experimental mouse tumor model, we generated a recombinant adenovirus expressing the E2 protein. Results: The E2 protein inhibited the growth of SiHa and BMK-16/myc cell lines, and repressed the E6 and E7 oncogenes. Moreover, the E2 protein induced DNA fragmentation and apoptosis through activation of caspases 8 and 3 in BMK-16/myc cells. On the other hand, E2 also showed antitumor effects in vivo. Conclusions: Our findings indicate that E2 exerts pro-apoptotic activity in a murine HPV-transformed cell line as well as an antitumor effect in vivo.

Effect of 60 Hz electromagnetic fields on the activity of hsp70 promoter: An in vitro study

We have evaluated the effect of 60 Hz sinusoidal magnetic fields (MF) at 8 and 8 μT on expression of the luciferase gene contained in a gene construct labelled as Electromagnetic Field‐plasmid (pEMF). The vector included the hsp70 promotor containing the 3 nCTCTn sequences previously described for the induction of hsp70 expression by magnetic fields, as well as the reporter of the luciferase gene. We also replicated the study of Lin et al. [Lin H, Blank M, Rossol‐Haseroth K, Goodman R. Regulating genes with electromagnetic response elements. J Cell Biochem 2001;81(1):143–48]. The pEMF plasmid was transfected into HeLa and BMK16 cell lines that were later exposed to either MF or thermal shock (TS). An increased luciferase expression was found in both the cells exposed to MF and TS compared with their control groups (P < 0.05). Furthermore, the combined effect of MF and TS was also analyzed. A synergistic effect between two factors was observed for this co‐exposure condition in terms of luciferase gene expression.