Detection of ALDH1 activity in rabbit hepatic VX2 tumors and isolation of ALDH1 positive cancer stem cells

Abstract Background Aldehyde dehydrogenase 1 (ALDH1) activity has been implicated in the therapeutic drug resistance of many malignancies and has been widely used as a marker to identify stem-like cells, including in primary liver cancer. Cancer stem cells (CSCs) are thought to play a crucial role in cancer progression and metastasis. In order to clarify the validity of the rabbit VX2 liver cancer model, we questioned if it expresses ALDH1 as a potential marker of CSCs. Hepatocellular carcinoma is a common malignancy worldwide and has poor prognosis. Most of the animal models used to study hepatocellular carcinoma are rodent models which lack clinical relevance. The rabbit VX2 model is a large animal model useful for preclinical and for developing drugs targeting cancer stem cells. Materials and methods We used flow cytometry to identify rabbit VX2 liver tumor cells that express ALDH1A1 activity at a high level and confirmed the results with RT-PCR, immunohistochemical and western blot analyses. Further, mRNA and protein expression analysis of tumor samples also express the markers for stemness like klf4, oct3/4, CD44 and nanog as well as the differentiation marker α-fetoprotein. Results We used Aldefluor flow cytometry-based assay to identify cells with high ALDH1 activity in the rabbit VX2 liver cancer model. We used the brightest 4.39 % of the total cancer cell population in our study. We performed semi-quantitative as well as real time PCR to characterize the stemness derived from VX2 tumors and tissues from normal rabbit liver. We demonstrated that VX2 tumors have higher expression of cancer stem cell markers such as AlDH1A1 and CD44 in comparison to normal rabbit liver cells. Additionally, real time PCR analysis of the same samples using syber-green demonstrated the significant change (p > 0.05) in the expression of genes. We validated the gene expression of the stemness markers by performing western blot and immunofluorescence. We showed that cancer stem cell markers (AlDH1A1, CD44) and the differentiation marker α-fetoprotein were upregulated in VX2 tumor cells. The same extent of upregulation was observed in stemness markers (klf4, oct3/4 and nanog) in VX2 tumors in comparison to normal rabbit liver. Conclusion The overall results of this study indicate that ALDH1 is a valid CSC marker for VX2 cancer. This finding suggests that the rabbit VX2 liver cancer model is useful in studying drug resistance in hepatocellular carcinoma and may be useful for basic and preclinical studies of other types of human cancer.http://deepblue.lib.umich.edu/bitstream/2027.42/117332/1/12967_2016_Article_785.pd

The Lyot project: toward exoplanet imaging and spectroscopy

Among the adaptive optics systems available to astronomers, the US Air Force Advanced Electro-Optical System (AEOS) is unique because it delivers very high order wave front correction. The Lyot Project includes the construction and installation of the world’s first diffraction-limited, optimized coronagraph that exploits the full astronomical potential of AEOS and represents a critical step toward the long-term goal of directly imaging and studying extrasolar planets (a.k.a. “exoplanets”). We provide an update on the Project, whose coronagraph saw first light in March 2004. The coronagraph is operating at least as well as predicted by simulations, and a survey of nearby stars has begun

Os saberes a ensinar e para ensinar matemática e suas relações com o ensino industrial brasileiro

Disponível em: http://revistas.upel.edu.ve/index.php/paradigma/article/view/6907/0Este trabalho é parte de uma pesquisa de doutorado que se preocupa em investigar o ensino de matemática no Liceu e na Escola Industrial de Florianópolis no período de 1937 a 1961. O objetivo é identificar os saberes a ensinar e para ensinar nos cursos do Liceu Industrial e da Escola Industrial de Florianópolis e quais as principais mudanças no ensino de Mat emática destas instituições de ensino industrial. Foram utilizados como referenciais teóricos Chervel (1990) sobre história das disciplinas escolares, Julia (2001) sobre cultura escolar , Hofstetter & Valente (2017) a respeito dos saberes a ensinar , Hofstet ter et al . (2017) na definição dos experts e Cellard (2008) para a análise documental . Percebeu - se que o ensino de Matemática era voltado para cálculos básicos, pois o enfoque era formar o aluno para a indústria, apesar de se intensificar o ensino de Álgeb ra e Trigonometria no início dos anos de 1940. Palavras - chave: Ensino Industrial. História da Educação Matemática. Experts . Saberes a ensinar. Saberes para ensinar

The Lyot project: toward exoplanet imaging and spectroscopy

Among the adaptive optics systems available to astronomers, the US Air Force Advanced Electro-Optical System (AEOS) is unique because it delivers very high order wave front correction. The Lyot Project includes the construction and installation of the world’s first diffraction-limited, optimized coronagraph that exploits the full astronomical potential of AEOS and represents a critical step toward the long-term goal of directly imaging and studying extrasolar planets (a.k.a. “exoplanets”). We provide an update on the Project, whose coronagraph saw first light in March 2004. The coronagraph is operating at least as well as predicted by simulations, and a survey of nearby stars has begun

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy

Oil-in-Water Nanoemulsions as Mucosal Vaccine Adjuvants: Characterization, Mechanism, Formulation, and Development of a Nanoemulsion-Based Burkholderia Cenocepacia Vaccine.

Surface active oil-in-water nanoscale emulsions have been developed as mucosal vaccine adjuvants capable of producing robust systemic, mucosal, and cellular immune responses against diverse microbial and recombinant antigenic proteins. This dissertation examines the development of nanoemulsion (NE) as a new generation nasopharyngeal adjuvant. Part of the thesis is organized to address the characterization of NE-induced immune response and includes the pre-clinical studies of a novel NE-based recombinant hepatitis B vaccine (HBsAg-NE). Our results suggest that nasal immunization with HBsAg-NE may be a safe and effective hepatitis B vaccine. The adjuvant induces specific IgG, mucosal IgA, and a Th1-biased cellular immunity. Immunogenicity is comparable to the standard alum-based vaccine. HBsAg-NE is stable for months at elevated temperatures because of the physical association of NE with the antigen and its stability was enhanced with buffered salt diluents. We also report that NE-based vaccines do not require specially engineered delivery devices. The prolonged stability and ease of delivery are direct advantages for use of NE-based vaccines in developing populations. We also evaluate the mechanism of NE adjuvant activity. NE promotes antigen internalization in nasal epithelium and loading into mucosal dendrtici cells (DC). Trafficking of the antigen to the submandibular lymph nodes and thymus occurs within 24 hours of intranasal vaccination. Administration of NE was not associated with the typical induction of local inflammation or histopathological changes. Microarray analysis shows the upregulation of only 1.6% of genes responsible for the production of acute phase inflammatory cytokines including IL6. Hallmark inflammatory cytokines such as IL4, and INF-γ were not measured in nasal secretions. The role of IL6 in NE adjuvant activity was examined by evaluating immunogenicity in IL6 mutant mice. The final component of the dissertation addresses the development of a NE-based Burkholderia cenocepacia outer membrane protein (OMP) vaccine. We demonstrate that NE is as a strong mucosal adjuvant for OMP and OMP-NE protects against experimental lung infections in mice. Overall, these findings confirm that NE is an excellent mucosal stimulant and support the further development of nanoemulsions as nasopharyngeal adjuvants. We conclude that nanoemulsion exhibits all the major desired characteristics of an adjuvant.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62434/1/pmakidon_1.pd

Mucosal Immunization with a Novel Nanoemulsion-Based Recombinant Anthrax Protective Antigen Vaccine Protects against Bacillus anthracis Spore Challenge▿

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ∼1,000 times the 50% lethal dose (∼1,000× LD50) of B. anthracis Ames strain spores (1.38 × 103 spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10× LD50 and 100× LD50 (1.2 × 106 and 1.2 × 107) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine

In Vitro Activities of a Novel Nanoemulsion against Burkholderia and Other Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species▿

Respiratory tract infection, most often involving opportunistic bacterial species with broad-spectrum antibiotic resistance, is the primary cause of death in persons with cystic fibrosis (CF). Species within the Burkholderia cepacia complex are especially problematic in this patient population. We investigated a novel surfactant-stabilized oil-in-water nanoemulsion (NB-401) for activity against 150 bacterial isolates recovered primarily from CF respiratory tract specimens. These specimens included 75 Burkholderia isolates and 75 isolates belonging to other CF-relevant species including Pseudomonas, Achromobacter, Pandoraea, Ralstonia, Stenotrophomonas, and Acinetobacter. Nearly one-third of the isolates were multidrug resistant, and 20 (13%) were panresistant based on standard antibiotic testing. All isolates belonging to the same species were genotyped to ensure that each isolate was a distinct strain. The MIC90 of NB-401 was 125 μg/ml. We found no decrease in activity against multidrug-resistant or panresistant strains. MBC testing showed no evidence of tolerance to NB-401. We investigated the activity of NB-401 against a subset of strains grown as a biofilm and against planktonic strains in the presence of CF sputum. Although the activity of NB-401 was decreased under both conditions, the nanoemulsion remained bactericidal for all strains tested. These results support NB-401's potential role as a novel antimicrobial agent for the treatment of infection due to CF-related opportunistic pathogens