Antibiofilm and antimicrobial activity of curcumin-chitosan nanocomplexes and trimethoprim-sulfamethoxazole on Achromobacter, Burkholderia, and Stenotrophomonas isolates.

BACKGROUND: Non-fermenting Gram-negative Achromobacter xylosoxidans, Burkholderia cepacia complex, and Stenotrophomonas maltophilia species cause healthcare-associated infections, often showing resistance to first-line drugs such as trimethoprim-sulfamethoxazole (TMP-SXT). The aim of this study was to determine the effect of curcumin-chitosan nanocomplexes on biofilm-producing clinical isolates of non-fermenting Gram-negative bacilli. METHODS: A. xylosoxidans, B. cepacia complex, and S. maltophilia clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility was determined by broth microdilution. Curcumin (Cur), chitosan (Chi), and sodium tripolyphosphate (TPP) were encapsulated by ionotropic gelation in magnetic nanoparticles (MNP) and were assessed by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR). Biofilm inhibition and eradication by Cur-Chi-TPP-MNP with TMP-SXT was assessed. RESULTS: Cur-Chi-TPP-MNP in combination with TMP-SXT showed biofilm inhibition activity in A. xylosoxidans (37.5 µg/mL), B. cepacia (18.75 µg/mL), and S. maltophilia (4.69-18.75 µg/mL) and low biofilm eradication activity in all three strains (150 - 300 µg/mL). CONCLUSIONS: Cur-Chi-TPP-MNP in combination with TMP-SXT was able to inhibit biofilm and in lower effect to eradicate established biofilms of clinical isolates of A. xylosoxidans, B. cepacia complex, and S. maltophilia species. Our results highlight the need to assess these potential treatment options to be used clinically in biofilm-associated infections.

Promoter Activation with Electromagnetism.

Gene expression can be modified by physical factors, such as heat, electricity and magnetic fields , and several types of ionizing and non-ionizing radiation. Promoter activation with extremely low-frequency electromagnetic fields is possible with an appropriate promoter, containing electromagnetic field response elements. Here, we describe how to examine promoter activation with extremely low-frequency electromagnetic fields, and we provide a step-by-step guide to the assembly of a solenoid suitable for promoter activation.

Alterations of Immune Parameters on Trichoplusia ni (Lepidoptera: Noctuidae) Larvae Exposed to Extremely Low-Frequency Electromagnetic Fields.

Worldwide mobile telephone and microwave use have resulted in an increasing presence of extremely low-frequency electromagnetic field radiations (ELF-EMFs) in ecosystems. ELF-EMFs have been associated with altered physiological processes that can adversely affect exposed organisms. In this study, Trichoplusia ni Hübner larvae were exposed for 24, 48, or 72 h to ELF-EMFs (60 Hz and 2.0 mT) to assess effects on immune response parameters and fertility. Trichoplusia ni life cycle and fertility were not affected by 24-h exposure. However, the number of apoptotic-like cells and cellular immune response significantly increased (P < 0.01) after 72-h exposure (2- and 1.1-fold, respectively), whereas hemolymph total protein and hemocyte cells were reduced (P < 0.01; 16 and 50%, respectively) after 48-h exposure. Hemocyte cell type analysis resulted in significantly (P < 0.01) higher granulocytes number in the unexposed (2-fold increase) and oenocytoids in the 72-h-exposed larvae (28.6-fold increase). Quantitative retrotranscription (RT-qPCR) showed that after 72-h ELF-EMF exposure, the antimicrobial peptides cecropin, lysozyme, gallerimycin, and pgrp were downregulated by 24,866.0, 2.69-, 119.1-, and 1.45-fold, respectively, whereas attacin and defensin were upregulated by 1.59- and 1.85-fold, respectively. The effect of ELF-EMFs on the T. ni larvae immune response and their potential impact on its physiology and susceptibility to pathogens are discussed. This information may provide new insight of ELF-EMFs on other pest species, as well as for the preservation of ecologically important species.

Systemic delivery and activation of the TRAIL gene in lungs, with magnetic nanoparticles of chitosan controlled by an external magnetic field.

Recently, functional therapies targeting a specific organ without affecting normal tissues have been designed. The use of magnetic force to reach this goal is studied in this work. Previously, we demonstrated that nanocarriers based on magnetic nanoparticles could be directed and retained in the lungs, with their gene expression under the control of a promoter activated by a magnetic field. Magnetic nanoparticles containing the TRAIL gene and chitosan were constructed using the ionic gelation method as a nanosystem for magnetofection and were characterized by microscopy, ζ-potential, and retention analysis. Magnetofection in the mouse melanoma cell line B16F10 in vitro induced TRAIL-protein expression and was associated with morphological changes indicative of apoptosis. Systemic administration of the nanosystem in the tail vein of mice with melanoma B16F10 at the lungs produced a very significant increase in apoptosis in tumoral cells that correlated with the number of melanoma tumor foci observed in the lungs. The high levels of apoptosis detected in the lungs were partially related to mouse survival. The data presented demonstrate that the magnetofection nanosystem described here efficiently induces apoptosis and growth inhibition of melanoma B16F10 in the lungs. This new approach for systemic delivery and activation of a gene based in a nanocomplex offers a potential application in magnetic gene delivery for cancer.

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 1980's 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.

CD3zeta expression and T cell proliferation are inhibited by TGF-beta1 and IL-10 in cervical cancer patients.

INTRODUCTION: Cervical 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 CD3zeta expression in peripheral blood and tumor-infiltrating T lymphocytes from women with squamous intraepithelial lesions (SIL) or cervical cancer (CC). RESULTS AND DISCUSSION: T 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-gamma were observed in women with CC. Moreover, infiltrating cells showed marginal responses. We also found that CD3zeta mRNA expression, whose protein is required for T cell activation, correlated with a decreased proliferation in advanced stages of the disease. CONCLUSION: Experiments with T cells from healthy donors in the presence TGF-beta1 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 vitro study.

We have evaluated the effect of 60 Hz sinusoidal magnetic fields (MF) at 8 and 8 microT 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.

Correlation between IL-10 gene expression and HPV infection in cervical cancer: a mechanism for immune response escape.

The present study was performed to determine IL-10 expression in cervical tissues in Mexican women according to the severity of the malignity and its association with HPV infection. IL-10 expression showed a clear tendency to increase during the different cervical cancer stages: 37% in LGSIL; 62% in HGSIL; and 84% in cancer. However, all the patients that expressed IL-10 were HPV positives; we found an association with HPV 16. These results suggest a clear relationship between IL-10, HPV and the stage of cervical cancer disease; this event could contribute to the immunosuppressive micro-environment in the tumor site.

Entamoeba invadens: influence of 60 Hz magnetic fields on growth and differentiation.

Exposure to extremely low-frequency (ELF) electromagnetic fields appears to result in a number of important biological changes. In the present study, we evaluated the effects of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5 and 2.0 mT on growth and differentiation of the protozoan Entamoeba invadens. We demonstrated an inhibitory growth effect when trophozoite cultures were exposed to 1.5 and 2.0 mT. Furthermore, we found that there was not a synergistic effect in cultures co-exposed to MF and Metronidazole, a cytotoxic drug against amoebic cells. In addition, MF exposure inhibited the encystation process of E. invadens.

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.

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.

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 (CWA(M6)). This hybrid precursor was produced but not cell-wall anchored in Lb. plantarum. We thus replaced CWA(M6) 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.

An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci.

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. To date, vaccine strategies against HPV-16 are based on the ability of the E7 oncoprotein to elicit an immune response against this virus. In this study, the use of an inducible or a constitutive system to produce the HPV-16 E7 protein in Lactococcus lactis, a non-pathogenic and non-invasive Gram-positive bacterium, was compared. The highest E7 production was obtained with the inducible system. When mice were immunized intranasally with recombinant lactococci expressing either inducible or constitutive E7, an antigen-specific cellular response (i.e. secretion of IL2 and IFN-gamma cytokines) was evoked and was substantially higher in mice receiving L. lactis expressing E7 with the inducible system. As bacterial antigen location may influence the immune response, recombinant L. lactis strains that produced E7 in three cellular locations, intracellular, secreted or cell-wall-anchored were evaluated. The highest immune response was elicited by administration of L. lactis producing an inducible cell-wall-anchored form of E7 protein. These promising results represent a step towards the development of a new, safe mucosal vector to treat HPV-related cervical cancer.