Biofilm Eradication and Inhibition of Methicillin-resistant Staphylococcus Clinical Isolates by Curcumin-Chitosan Magnetic Nanoparticles.

Biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococci (MR-CoNS) are a clinical challenge for the treatment of healthcare-associated infections. As alternative antimicrobial options are needed, we aimed to determine the effect of curcumin-chitosan magnetic nanoparticles on the biofilm of staphylococcal clinical isolates. MRSA and CoNS clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing was performed by broth microdilution. Nanoparticles were synthesized by co-precipitation of magnetic nanoparticles (MNP) and encapsulation by ionotropic gelation of curcumin (Cur) and chitosan (Chi). Biofilm inhibition and eradication by nanoparticles with and without the addition of oxacillin was assessed on staphylococcal strains. Cur-Chi-MNP showed antimicrobial activity on planktonic cells of MRSA and MR-CoNS strains and inhibited biofilm of MRSA. The addition of OXA to Cur-Chi-MNP increased biofilm inhibition and eradication activity against all Staphylococci strains (p=0.0007); higher biofilm activity was observed in early biofilm stages. Cur-Chi-MNP showed antimicrobial and biofilm inhibition activity against S. aureus. The addition of OXA increased biofilm inhibition and eradication activity against all Staphylococci strains. A combination treatment of Cur-Chi-MNP and OXA could be potentially used to treat staphylococcal biofilm-associated infections in its early stages before the establishment of biofilm bacterial cells.

CAR-T Cell Therapy: From the Shop to Cancer Therapy.

Cancer is a worldwide health problem. Nevertheless, new technologies in the immunotherapy field have emerged. Chimeric antigen receptor (CAR) technology is a novel biological form to treat cancer; CAR-T cell genetic engineering has positively revolutionized cancer immunotherapy. In this paper, we review the latest developments in CAR-T in cancer treatment. We present the structure of the different generations and variants of CAR-T cells including TRUCK (T cells redirected for universal cytokine killing. We explain the approaches of the CAR-T cells manufactured ex vivo and in vivo. Moreover, we describe the limitations and areas of opportunity for this immunotherapy and the current challenges of treating hematological and solid cancer using CAR-T technology as well as its constraints and engineering approaches. We summarize other immune cells that have been using CAR technology, such as natural killer (NK), macrophages (M), and dendritic cells (DC). We conclude that CAR-T cells have the potential to treat not only cancer but other chronic diseases.

Potential of Nanocellulose as a Dietary Fiber Isolated from Brewer's Spent Grain.

Steady growth in beer production is increasing the number of by-products named brewers' spent grain. Such by-products are a source of several components, where cellulose is usually present in high amounts. The aim of this study was to develop a protocol to obtain a mix of cellulose microfibers with an average diameter of 8-12 µm and cellulose nanoplatelets with an average thickness of 100 nm, which has several applications in the food industry. The process comprised one alkaline treatment followed by acid hydrolysis, giving a new mix of micro and nanocellulose. This mix was characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and laser scanning microscopy corroborating the presence and measurements of the cellulose nanostructure, showing an aspect ratio of up to 500. Finally, we demonstrated that the administration of this new type of nanocellulose allowed us to control the weight of mice (feed intake), showing a significant percentage of weight reduction (4.96%) after 15 days compared with their initial weight, indicating the possibility of using this material as a dietary fiber.

Systemic Delivery of Magnetogene Nanoparticle Vector for Gene Expression in Hypoxic Tumors.

Cancer is a disease that causes millions of deaths per year worldwide because conventional treatments have disadvantages such as unspecific tumor selectivity and unwanted toxicity. Most human solid tumors present hypoxic microenvironments and this promotes multidrug resistance. In this study, we present "Magnetogene nanoparticle vector" which takes advantage of the hypoxic microenvironment of solid tumors to increase selective gene expression in tumor cells and reduce unwanted toxicity in healthy cells; this vector was guided by a magnet to the tumor tissue. Magnetic nanoparticles (MNPs), chitosan (CS), and the pHRE-Luc plasmid with a hypoxia-inducible promoter were used to synthesize the vector called "Magnetogene nanoparticles" by ionic gelation. The hypoxic functionality of Magnetogene vector nanoparticles was confirmed in the B16F10 cell line by measuring the expression of the luciferase reporter gene under hypoxic and normoxic conditions. Also, the efficiency of the Magnetogene vector was confirmed in vivo. Magnetogene was administered by intravenous injection (IV) in the tail vein and directed through an external magnetic field at the site of tumor growth in C57Bl/6 mice. A Magnetogene vector with a size of 50 to 70 nm was directed and retained at the tumor area and gene expression was higher at the tumor site than in the others tissues, confirming the selectivity of this vector towards hypoxic tumor areas. This nanosystem, that we called the "Magnetogene vector" for systemic delivery and specific gene expression in hypoxic tumors controlled by an external magnetic designed to target hypoxic regions of tumors, can be used for cancer-specific gene therapies.

In Planta Detection of Beauveria bassiana (Ascomycota: Hypocreales) Strains as Endophytes in Bean (Phaseolus vulgaris L.).

Beauveria bassiana (B. bassiana) is a significant entomopathogenic fungus (EPF) in agriculture as a sprayable biocontrol agent. It has the potential to be established as an endophyte (ENP) in various crops, resulting in beneficial effects for the host plants, including resistance to pest insects and increased growth and yield. However, it is not known whether a B. bassiana strain has such a favorable impact on the plant, since it is a common soil microorganism. Therefore, techniques that allow strain monitoring will be advantageous. To date, methods for detecting or monitoring a specific EPF strain after external application are scarce. In the present study, an in planta nested PCR technique was standardized to differentiate between three B. bassiana strains (GHA, PTG4, and BB37) established as endophytes in bean plants under laboratory conditions by detecting the insertion profile of four group I introns located in the 28S gene of B. bassiana ribosomal DNA. This technique recognized a distinct pattern of bands of different sizes for each strain, with a sensitivity of 1 pg per 10 ng of plant DNA. This molecular approach may be more effective monitoring B. bassiana strains after application to evaluate their significance on crops.

Selenite Downregulates STAT3 Expression and Provokes Lymphocytosis in the Liver of Chronically Exposed Syrian Golden Hamsters.

Arsenic is considered a worldwide pollutant that can be present in drinking water. Arsenic exposure is associated with various diseases, including cancer. Antioxidants as selenite and α-tocopherol-succinate have been shown to modulate arsenic toxic effects. Since changes in STAT3 and PSMD10 gene expression have been associated with carcinogenesis, the aim of this study was to evaluate the effect of arsenic exposure and co-treatments with selenite or α-tocopherol-succinate on the expression of these genes, in the livers of chronically exposed Syrian golden hamsters. Animals were divided into six groups: (i) control, (ii) chronically treated with 100 ppm arsenic, (iii) treated with 6 ppm α-tocopherol-succinate (α-TOS), (iv) treated with 8.5 ppm selenite, (v) treated with arsenic + α-TOS, and (vi) treated with arsenic + selenite. Urine samples and livers were collected after 20 weeks of continuous exposure. The urine samples were analyzed for arsenic species by atomic absorption spectrophotometry, and real-time RT-qPCR analysis was performed for gene expression evaluation. A reduction in STAT3 expression was observed in the selenite-treated group. No differences in PSMD10 expression were found among groups. Histopathological analysis revealed hepatic lymphocytosis in selenite-treated animals. As a conclusion, long-term exposure to arsenic does not significantly alter the expression of STAT3 and PSMD10 oncogenes in the livers of hamsters; however, selenite down-regulates STAT3 expression and provokes lymphocytosis.

Association study of genetic polymorphisms in proteins involved in oseltamivir transport, metabolism, and interactions with adverse reactions in Mexican patients with acute respiratory diseases.

Oseltamivir, a pro-drug, is the best option for treatment and chemoprophylaxis for influenza outbreaks. However, many patients treated with oseltamivir developed adverse reactions, including hypersensitivity, gastritis, and neurological symptoms. The aim of this study was to determine the adverse drug reactions (ADRs) in Mexican patients treated with oseltamivir and whether these ADRs are associated with SNPs of the genes involved in the metabolism, transport, and interactions of oseltamivir. This study recruited 310 Mexican patients with acute respiratory diseases and treated them with oseltamivir (75 mg/day for 5 days) because they were suspected to have influenza A/H1N1 virus infection. Clinical data were obtained from medical records and interviews. Genotyping was performed using real-time polymerase chain reaction and TaqMan probes. The association was assessed under genetic models with contingency tables and logistic regression analysis. Out of 310 patients, only 38 (12.25%) presented ADRs to oseltamivir: hypersensitivity (1.9%), gastritis (10%), and depression and anxiety (0.9%). The polymorphism ABCB1-rs1045642 was associated with adverse drug reactions under the recessive model (P = 0.017); allele C was associated with no adverse drug reactions, while allele T was associated with adverse drug reactions. The polymorphisms SLC15A1-rs2297322, ABCB1-rs2032582, and CES1-rs2307243 were not consistent with Hardy-Weinberg equilibrium, and no other associations were found for the remaining polymorphisms. In conclusion, the polymorphism rs1045642 in the transporter encoded by the ABCB1 gene is a potential predictive biomarker of ADRs in oseltamivir treatment.

The novel immunomodulator IMMUNEPOTENT CRP combined with chemotherapy agent increased the rate of immunogenic cell death and prevented melanoma growth.

Immunogenic cell death is a cell death modality that stimulates the immune system to combat cancer cells. IMMUNEPOTENT CRP (ICRP) is a mixture of substances of low molecular weight obtained from bovine spleens that exhibits in vitro cytotoxic activity on different tumor cell lines and modulates the immune response in vivo. The aim of the present study was to determine whether the cytotoxic effect of ICRP and its combination with oxaliplatin (OXP) on murine melanoma B16F10 cells was due to immunogenic cell death. The cytotoxic assay was performed using flow cytometry to detect Annexin V and propidium iodide staining, and calreticulin (CRT) exposure. Adenosine triphosphate, heat shock protein (HSP) 70, HSP90 and high mobility group box 1 (HMGB1) release were identified using bioluminescence, western blot and ELISA assays, respectively. The present in vitro study demonstrated that treatments with ICRP or OXP induced cell death in a time-dependent manner, but treatment with the combination of ICRP + OXP increased the cytotoxic effect following 24 h of treatment. CRT exposure and release of adenosine triphosphate (ATP), HSP70, HSP90 and HMGB1 were induced by treatment with ICRP, and the combination of ICRP + OXP increased the exposure and release of damage-associated molecular patterns (DAMPs), while OXP treatment only induced CRT exposure, ATP and HMGB1 release. The in vivo experiments demonstrated that administration of tumor-derived DAMP-rich cell lysates derived from B16F10 cells treated with ICRP and the combination of ICRP + OXP prevented melanoma growth; however, OXP treatment did not. These results suggested that IMMUNEPOTENT CRP may be used as an agent to increase the ability of antitumor drugs to induce immunogenic cell death and prevent the growth of melanoma.

In vivo Antitumor Effect of an HPV-specific Promoter driving IL-12 Expression in an HPV 16-positive Murine Model of Cervical Cancer.

Human papillomavirus (HPV) is a DNA virus that infects epithelial cells and has been implicated in the development of cervical cancer. Few therapeutic strategies have been designed for the treatment of cervical intraepithelial neoplasia, a precursor of cervical cancer. In these early stages, the HPV E2 protein is the most important viral factor involved in viral gene expression and plays crucial roles during the vegetative viral cycle in epithelial cells. Papillomavirus E2 binds specifically to palindromic ACCN6GGT sequences, referred to as the E2 binding sites (E2BS), which are concentrated within the viral long control region, and which are responsible for regulation of the HPV protein's expression. Here, we consider E2BS as a candidate sequence to induce the expression of antiviral therapeutic genes selectively in HPV-infected cells expressing the E2 protein. This study focuses on the use of an HPV-specific promoter comprised of four E2BS to drive the expression of IL-12, leading to an antitumor effect in an HPV-positive murine tumor model. The therapeutic strategy was implemented via viral gene therapy using adenoviral vectors with recombinant E2 and IL-12 genes and E2BS-IL-12. We demonstrate that the HPV-specific promoter E2BS is functional in vitro and in vivo through transactivation of HPV E2 transcription factor.

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

BACKGROUND: Colloidal 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. METHODS: MCF-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. RESULTS: Colloidal 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. CONCLUSIONS: The present results showed that colloidal silver might be a potential alternative agent for human breast cancer therapy.

Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells.

It has been reported that 50-60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 µT on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line.

Expression of DC-SIGN in peripheral blood dendritic cells of patients with typical, slow, and rapid progression to AIDS.

The main access route for human immunodeficiency virus (HIV) into the lymph nodes is through the mucosa. Once there, dendritic cells (DCs) are the first cells to interact with the virus. Then, DCs can uptake and transport to the lymph nodes, beginning a disseminated infection. Interaction between the virus and DCs is mediated by the receptor DC-SIGN. This study seeks to determine any relationship between HIV-AIDS immunopathology and DC-SIGN expression levels in DCs from typical, rapid, and slow progressors. A DC separation system was implemented using peripheral blood mononuclear cells from infected subjects. The study included 27 patients classified as typical, rapid, and slow progressors according to their clinical and epidemiological files. Finally, quantification of DC-SIGN was achieved by real-time PCR and by applying the Relative Quantification Scheme (DeltaDeltaCt). We isolated DCs from peripheral blood of 27 HIV-infected patients. Nineteen were considered as typical progressors, five as slow progressors, and three as rapid progressors. No significant differences were observed on the expression levels of DC-SIGN among the three groups of patients. Even if there are differences in expression levels among the analyzed patients, we did not find any significant differences in DC-SIGN expression among the three included groups. We therefore cannot conclude that the expression level of the receptor is related with the progression to AIDS.

Association between the expression of IL-10 and T cell activation proteins loss in early breast cancer patients.

Breast cancer patients may express abnormal cellular immune responses affecting their immunological competence. The analysis of immunological parameters may be useful as indicators of T cell function. To determine the expression of lymphocyte activation proteins and cytokines in tumor and non-metastatic axillary lymph nodes, 30 breast cancer patients were monitored. CD3 polypeptides, PTKs (protein tyrosine kinases) and phosphorylated tyrosines were studied by Western Blot and cytokines mRNA expression was determined by RT-PCR (reverse transcription-polymerase chain reaction). This group of patient had shown high immunohistochemistry expression of IL-10 in tumors. Activation proteins were mainly expressed in involved lymph nodes comparing with their expression in tumors. The differences in expression of CD3 polypeptides and p56(lck) between both locations were significant. There was no statistical association between PTKs and IL-10 in the tumor but more than 50% of cases who express IL-10 lost p56(lck), p59(fyn). A direct association between IL-10 and CD3-polypeptides was observed, however 52.2% of patients who express IL-10 did not express 41 kDa CD3-zeta form. IL-10 mRNA was detected in more than 50% of tumors contrary to the prevalence of type 1 cytokines in regional nodes (40%). The lack of expression of lymphocyte activations proteins and the high expression of IL-10 suggest a downregulation on T cells function in the tumors. These results are useful in order to understand the local immune response that would be key in the control of the tumor progression.

In vivo expression of immunosuppressive cytokines in human papillomavirus-transformed cervical cancer cells.

Genital human Papillomavirus infection is common and only a minor fraction of infected subjects develop progressing cervical epithelial lesions or cancer. Bypassing local immune responses is important for the development of cervical cancer. In this work we determined the cytokine pattern in samples from patients with cervical cancer. Thus, we examined the local mRNA expression profile of helper T cell type 1 (Th1), Th2, and Th3 cytokines in HPV-positive cervical cancer biopsies by reverse transcription-polymerase chain reaction. Our data indicate that 80% of the tumors expressed low levels of CD4 mRNA, with all of them expressing higher CD8 mRNA levels. Most tumors expressed interleukin (IL)-4 and IL-10 mRNAs and, most importantly, all of them expressed transforming growth factor (TGF)-beta1 and interferon gamma mRNA. None of the tumors studied expressed IL-12, IL-6, or tumor necrosis factor (TNF) mRNA. Immunohistochemical analysis identified IL-10 only in tumor cells and koilocytic cells, but not in tumor-infiltrating lymphocytes, suggesting that IL-10-producing cells are those transformed by HPV. We found a correlation between immunostaining for IL-10 protein and the level of IL-10 mRNA expression. Moreover, supernatants from HPV-transformed cell cultures contained IL-10 and TGF- beta1. Our findings indicate a predominant expression of immunosuppressive cytokines, which might help downregulate tumor-specific immune responses in the microenvironment of the tumor. This information may be useful for cervical cancer immunotherapies or for therapeutic vaccine design against Human Papillomavirus.

Relationship between IL-10 and tumor markers in breast cancer patients.

Tumor markers are expressed due to molecular alterations of the tumor cells, and we can relate them to the immune system to find new associations to improve prognosis. IL-10 inhibits the generation of immune responses at the tumor site. To determine IL-10 expression in the tumor microenvironment and to associate it with certain tumor markers, 27 breast cancer patients were monitored by immunohistochemistry. The results showed that 23 breast cancer samples exhibited a strong expression of IL-10. IL-10 was associated with some poor prognosis tumor makers. A direct association between IL-10, Bcl-2, and Bax was detected. The relationship between IL-10 and Bax was statistically significant (P=0.001). An inverse association of IL-10 with p53 was observed. IL-10 reflects a suppressive tumor microenvironment, and its relationship with apoptosis markers can suggest an increase in the aggressiveness of the tumor even if it still is at an early stage.

Perspectivas para el desarrollo de vacunas e inmunoterapia contra cáncer cervicouterino / Perspectives for vaccines and immunotherapy againts cervical cancer

El cáncer cervicouterino representa un grave problema de salud pública, debido a la asociación de la neoplasia con el virus del papiloma humano; actualmente se realizan estudios usados estrategias dirigidas a combatir este patógeno, mediante vacunas, que podrían ser de gran utilidad para el control de la progresión de la enfermedad. El estudio tanto de la inmunología humoral como celular ha servido para el desarrollo de vcunas. Así, la utilización de partículas virales sintéticas para el estudio de anticuerpos neutralizantes y el uso de proteínas tempranas virales, entre otras, para la inducción de inmunidad mediada por células, han sido la pauta para realizar estudios que dirijan la respuesta inmune para prevenir la infección celular tanto hacia células infectadas no transformadas como hacia células transformadas viralmente con resultados favorables