College of Agriculture and Life Sciences (CALS)

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Antibiotics ameliorate lupus-like symptoms in mice
Mu, Qinghui; Tavella, Vincent J.; Kirby, Jay L.; Cecere, Thomas E.; Chung, Matthias; Lee, Jiyoung; Li, Song; Ahmed, Sattar Ansar; Eden, Kristin; Allen, Irving C. (Nature, 2017-10-20)
Gut microbiota and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of systemic lupus erythematosus (SLE) is unclear. Here we report that oral antibiotics given during active disease removed harmful bacteria from the gut microbiota and attenuated SLE-like disease in lupus-prone mice. Using MRL/lpr mice, we showed that antibiotics given after disease onset ameliorated systemic autoimmunity and kidney histopathology. They decreased IL-17-producing cells and increased the level of circulating IL-10. In addition, antibiotics removed Lachnospiraceae and increased the relative abundance of Lactobacillus spp., two groups of bacteria previously shown to be associated with deteriorated or improved symptoms in MRL/lpr mice, respectively. Moreover, we showed that the attenuated disease phenotype could be recapitulated with a single antibiotic vancomycin, which reshaped the gut microbiota and changed microbial functional pathways in a time-dependent manner. Furthermore, vancomycin treatment increased the barrier function of the intestinal epithelium, thus preventing the translocation of lipopolysaccharide, a cell wall component of Gram-negative Proteobacteria and known inducer of lupus in mice, into the circulation. These results suggest that mixed antibiotics or a single antibiotic vancomycin ameliorate SLE-like disease in MRL/lpr mice by changing the composition of gut microbiota.
Cell-free protein synthesis of norovirus virus-like particles
Sheng, Jiayuan; Lei, Shaohua; Yuan, Lijuan; Feng, Xueyang (Royal Society of Chemistry, 2017-05-25)
Norovirus vaccine development largely depends on recombinant virus-like-particles (VLPs). Norovirus VLPs have been produced in several cell-based expression systems with long production times. Here we report, for the first time, that norovirus VLPs can be expressed and assembled by using a cell-free protein expression system within four hours.
Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression Model
Creekmore, Amy L.; Silkworth, William T.; Cimini, Daniela; Jensen, Roderick V.; Roberts, Paul C.; Schmelz, Eva M. (PLOS, 2011-03-03)
Background Ovarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment. Methodology/Principal Findings Mouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII. Conclusions/Significance Our studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression.
Dehydroepiandrosterone protects vascular endothelial cells against apoptosis through a G alpha(i) protein-dependent activation of phosphatidylinositol 3-kinase/Akt and regulation of antiapoptotic Bcl-2 expression
Liu, D. M.; Si, H. W.; Reynolds, K. A.; Zhen, W.; Jia, Z. Q.; Dillon, J. S. (Endocrine Society, 2007-07)
The adrenal steroid dehydroepiandrosterone (DHEA) may improve vascular function, but the mechanism is unclear. In the present study, we show that DHEA significantly increased cell viability, reduced caspase-3 activity, and protected both bovine and human vascular endothelial cells against serum deprivation-induced apoptosis. This effect was dose dependent and maximal at physiological concentrations (0.1-10 nM). DHEA stimulation of bovine aortic endothelial cells resulted in rapid and dose-dependent phosphorylation of Akt, which was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), the upstream kinase of Akt. Accordingly, inhibition of PI3K or transfection of the cells with dominant-negative Akt ablated the antiapoptotic effect of DHEA. The induced Akt phosphorylation and subsequent cytoprotective effect of DHEA were dependent on activation of G alpha(i) proteins, but were estrogen receptor independent, because these effects were blocked by pertussis toxin but not by the estrogen receptor inhibitor ICI182,780 or the aromatase inhibitor aminoglutethimide. Finally, DHEA enhanced antiapoptotic Bcl-2 protein expression, its promoter activity, and gene transcription attributable to the activation of the PI3K/Akt pathway. Neutralization of Bcl-2 by antibody transfection significantly decreased the antiapoptotic effect of DHEA. These findings provide the first evidence that DHEA acts as a survival factor for endothelial cells by triggering the G alpha(i)-PI3K/Akt-Bcl-2 pathway to protect cells against apoptosis. This may represent an important mechanism underlying the vascular protective effect of DHEA.
Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis
Salmanzadeh, Alireza; Kittur, Harsha; Sano, Michael B.; Roberts, Paul C.; Schmelz, Eva M.; Davalos, Rafael V. (American Institute of Physics, 2012-06-01)
Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3699973] Actual pdf downloaded from NCBI.
Dietary epicatechin improves survival and delays skeletal muscle degeneration in aged mice
Si, Hongwei; Wang, Xiaoyong; Zhang, Longyun; Parnell, Laurence D.; Admed, Bulbul; LeRoith, Tanya; Ansah, Twum-Ampofo; Zhang, Lijuan; Li, Jianwei; Ordovas, Jose M.; Si, Hongzong; Liu, Dongmin; Lai, Chao-Qiang (2019-01)
We recently reported that epicatechin, a bioactive compound that occurs naturally in various common foods, promoted general health and survival of obese diabetic mice. It remains to be determined whether epicatechin extends health span and delays the process of aging. In the present study, epicatechin or its analogue epigallocatechin gallate (EGCG) (0.25% w/v in drinking water) was administered to 20-mo-old male C57BL mice fed a standard chow. The goal was to determine the antiaging effect. The results showed that supplementation with epicatechin for 37 wk strikingly increased the survival rate from 39 to 69%, whereas EGCG had no significant effect. Consistently, epicatechin improved physical activity, delayed degeneration of skeletal muscle (quadriceps), and shifted the profiles of the serum metabolites and skeletal muscle general mRNA expressions in aging mice toward the profiles observed in young mice. In particular, we found that dietary epicatechin significantly reversed age-altered mRNA and protein expressions of extracellular matrix and peroxisome proliferator-activated receptor pathways in skeletal muscle, and reversed the age-induced declines of the nicotinate and nicotinamide pathway both in serum and skeletal muscle. The present study provides evidence that epicatechin supplementation can exert an antiaging effect, including an increase in survival, an attenuation of the aging-related deterioration of skeletal muscles, and a protection against the aging-related decline in nicotinate and nicotinamide metabolism.Si, H., Wang, X., Zhang, L., Parnell, L. D., Admed, B., LeRoith, T., Ansah, T.-A., Zhang, L., Li, J., Ordovas, J. M., Si, H., Liu, D., Lai, C.-Q. Dietary epicatechin improves survival and delays skeletal muscle degeneration in aged mice.
Differentiating Vaccine-Related Fowl Cholera from Naturally Occurring Disease
Hutcheson, Anna R.; Thompson, Kasey; Maurer, John J.; Ferguson, Naola; Grogan, Karen; Roney, Stephen; Seahorn, Harmony; Lobsinger, Chris; Lee, Margie D. (2020-12)
Vaccine-related fowl cholera must be considered when flock mortality increases after use of a live Pasteurella multocida vaccine product. All registered live vaccines serotype as Heddleston 3,4; however, in some regions this is also the most common scrotype of outbreak isolates in broiler breeders and turkeys. Therefore, serotyping may not be useful for diagnosing vaccine-related fowl cholera. This project sought to apply a vaccine-specific test to differentiate vaccine-related disease from naturally occurring outbreaks. Results indicate that vaccine strains were commonly isolated from broiler breeders exhibiting signs of fowl cholera postvaccination, but some of these isolates exhibited only serotype 4 antigenicity. The isolates' lipopolysaccharides, the target antigen for serotyping, contained compositional changes that may explain the varying serotype results and virulence of the commercial preparations. These results suggest that vaccine-related disease may be common in broiler breeders, and live commercial vaccine preparations need to be assessed for serotype and titer prior to use in order to reduce vaccine-related fowl cholera.
Effect of hydrogen peroxide and other protease inhibitors on Cryptosporidium parvum excystation and in vitro development
Kniel, Kalmia E.; Sumner, Susan S.; Pierson, Merle D.; Zajac, Anne M.; Hackney, Cameron Raj; Fayer, Ronald; Lindsay, David S. (American Society of Parasitology, 2004-08)
This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by > 90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.
Effects of high pressure processing on infectivity of Toxoplasma gondii oocysts for mice
Lindsay, David S.; Collins, Marina V.; Jordan, C. N.; Flick, George J. Jr.; Dubey, Jitender P. (American Society of Parasitology, 2005-06)
High pressure processing (HPP) has been shown to be an effective non-thermal method of eliminating non-spore forming bacteria from a variety of food products. The shelf-life of the products is extended and the sensory features of the food are not or only minimally effected by HPP. The present study examined the effects of HPP using a commercial scale unit on the viability of Toxoplasma gondii oocysts. Oocysts were exposed from 100 to 550 MPa for I min in the HPP unit and then HPP treated oocysts were orally fed to groups of mice. Oocysts treated with 550 MPa or less did not develop structural alterations when viewed with light microscopy. Oocysts treated with 550 MPa, 480 MPa, 400 Mpa, or 340 MPa were tendered noninfectious for mice. Mice fed oocysts treated with no or 100 to 270 MPa became infected and most developed acute toxoplasmosis and were killed or died 7 to 10 days after infection. These results suggest that HPP technology may be useful in the removal of T. gondii oocysts from food products.
Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi
Jordan, C. N.; Zajac, Anne M.; Holliman, D.; Flick, George J. Jr.; Lindsay, David S. (American Society of Parasitology, 2005-12)
High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.
Effects of high-pressure processing on Toxoplasma gondii tissue cysts in ground pork
Lindsay, David S.; Collins, Marina V.; Holliman, D.; Flick, George J. Jr.; Dubey, Jitender P. (American Society of Parasitology, 2006-02)
Ingestion of Toxoplasma gondii tissue Cysts can result in severe disease in immunocompromised individuals and pregnant women. Treatment of mean and meat products to eliminate. viable T. gondii tissue cysts would provide a means to protect consumers. In this Study, we examined the effects of high-pressure processing (HPP) on ground pork containing viable tissue Cysts of the VEG strain of T. gondii. Ground pork containing tissue cysts was exposed to 400, 300, 200, 100, or 0 MPa treatment for 30, 60, or 90 sec in a commercial HPP unit. The HPP-treated ground pork Was subjected to acid-pepsin digestion and bioassayed in mice. The results of the mouse bioassay revealed that none of the mice inoculated with tissue cysts exposed to 400 or 300 MPa became infected, whereas all mice inoculated with tissue cysts exposed to 200, 100, or 0 MPa became infected with T. gondii regardless of exposure time. Results indicate that HPP treatment of ground pork with 300 MPa of pressure will render tissue cysts of T. gondii nonviable and make pork safe for human consumption.
EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity
Sun, Zhixiong; Xu, Xiguang; He, Jianlin; Murray, Alexander; Sun, Ming-an; Wei, Xiaoran; Wang, Xia; McCoig, Emmarose; Xie, Evan; Jiang, Xi; Li, Liwu; Zhu, Jinsong; Chen, Jianjun; Morozov, Alexei; Pickrell, Alicia M.; Theus, Michelle H.; Xie, Hehuang David (2019-08-29)
Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.
Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.
Rothschild, Daniel E.; Zhang, Yao; Diao, Na; Lee, Christina K.; Chen, Keqiang; Caswell, Clayton C.; Slade, Daniel J.; Helm, Richard F.; LeRoith, Tanya; Li, Liwu; Allen, Irving C. (2016-12-03)
Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m(-/-) mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m(-/-) animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m(-/-) mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m(-/-) mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.
Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation
Hendricks-Wenger, Alissa; Aycock, Kenneth N.; Nagai-Singer, Margaret A.; Coutermarsh-Ott, Sheryl; Lorenzo, Melvin F.; Gannon, Jessica; Uh, Kyungjun; Farrell, Kayla; Beitel-White, Natalie; Brock, Rebecca M.; Simon, Alexander; Morrison, Holly A.; Tuohy, Joanne L.; Clark-Deener, Sherrie; Vlaisavljevich, Eli; Davalos, Rafael V.; Lee, Kiho; Allen, Irving C. (Nature Research, 2021-04-07)
New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.
Evaluating the relationship between fecal egg count, FAMACHA score, and weight in dewormed and non-dewormed Katahdin rams during a parasite challenge
Galyon, Hailey R.; Zajac, Anne M.; Wright, D. Lee; Greiner, Scott P.; Bradford, Heather L. (Oxford University Press, 2020-10-01)
The objective of this study was to evaluate and to estimate the relationship between fecal egg counts (FECs) and FAMACHA score and the body weight of growing Katahdin rams during a parasite challenge. One of the largest factors negatively influencing reproduction and economics in the sheep industry is gastrointestinal nematode (GIN) parasites. Due to anthelmintic resistance of these parasites, animals are selected for parasite resistance using FEC and FAMACHA scores. Data were used from the Virginia Tech Southwest Agricultural Research and Extension Center Ram Test in Glade Spring, VA, from the year 2012 to 2018 in which animals were tested in 14-d intervals for 70 d. Mixed models for repeated weight measurements were made from backward stepwise selection to evaluate the relationships between weight and GIN FEC. A total of 576 animals within 23 contemporary groups derived from test year and pasture group were analyzed. Ram, contemporary group, and consignor were considered random effects, and fixed effects were birth type, test day, age, age squared, starting weight, FEC, and FAMACHA score. Pairwise contrasts were used in the statistical analysis of parameters and their interactions. Weight and age were found to have a quadratic relationship. Increased FEC was associated with weight loss at a rate of 0.00030 kg/FEC (P < 0.0001). Animals dewormed at any point during the trial weighed less than those that were not and increased with test day to a maximum difference of 4.66 kg (P < 0.001). FAMACHA score was found to be significant (P < 0.05), but a direct relationship with weight was not conclusive. Overall, rams with severe enough parasite load to require deworming had lesser weights, which could impact the profitability of sheep production and reinforced the need to select animals that had greater innate parasite resistance.
Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis
Zhang, Zhenlu; He, Guijuan; Han, Gil-Soo; Zhang, Jiantao; Catanzaro, Nicholas; Diaz, Arturo; Wu, Zujian; Carman, George M.; Xie, Lianhui; Wang, Xiaofeng (PLOS, 2018-04-12)
Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1 Delta), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1 Delta cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1 Delta cells, BMV replicated as efficiently as in pah1 Delta cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1 Delta cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.
In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters
Hu, Yun; Ehrich, Marion F.; Fuhrman, Kristel; Zhang, Chenming (Springer, 2014-08-27)
Due to the many beneficial properties combined from both poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and liposomes, lipid-PLGA hybrid NPs have been intensively studied as cancer drug delivery systems, bio-imaging agent carriers, as well as antigen delivery vehicles. However, the impact of lipid composition on the performance of lipid-PLGA hybrid NPs as a delivery system has not been well investigated. In this study, the influence of lipid composition on the stability of the hybrid NPs and in vitro antigen release from NPs under different conditions was examined. The uptake of hybrid NPs with various surface charges by dendritic cells (DCs) was carefully studied. The results showed that PLGA NPs enveloped by a lipid shell with more positive surface charges could improve the stability of the hybrid NPs, enable better controlled release of antigens encapsulated in PLGA NPs, as well as enhance uptake of NPs by DC.
Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency
Lei, Shaohua; Ryu, Junghyun; Wen, Ke; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Weiss, Mariah; Li, Guohua; Samuel, Helen; Clark-Deener, Sherrie; Jiang, Xi; Lee, Kiho; Yuan, Lijuan (Nature Publishing Group, 2016-04-27)
Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.
Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells
Salmanzadeh, Alireza; Sano, Michael B.; Gallo-Villanueva, R. C.; Roberts, Paul C.; Schmelz, Eva M.; Davalos, Rafael V. (American Institute of Physics, 2013-01-01)
In this study, the electrical properties of four different stages of mouse ovarian surface epithelial (MOSE) cells were investigated using contactless dielectrophoresis (cDEP). This study expands the work from our previous report describing for the first time the crossover frequency and cell specific membrane capacitance of different stages of cancer cells that are derived from the same cell line. The specific membrane capacitance increased as the stage of malignancy advanced from 15.39 +/- 1.54 mF m(-2) for a non-malignant benign stage to 26.42 +/- 1.22 mF m(-2) for the most aggressive stage. These differences could be the result of morphological variations due to changes in the cytoskeleton structure, specifically the decrease of the level of actin filaments in the cytoskeleton structure of the transformed MOSE cells. Studying the electrical properties of MOSE cells provides important information as a first step to develop cancer-treatment techniques which could partially reverse the cytoskeleton disorganization of malignant cells to a morphology more similar to that of benign cells. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4788921] Actual pdf downloaded from NCBI.
Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice
Bandara, Aloka B.; Champion, Anna E.; Wang, X.; Berg, G.; Apicella, Michael A.; McLendon, M.; Azadi, P.; Snyder, D. S.; Inzana, Thomas J. (PLOS, 2011-04-22)
Background: Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized. Methods and Findings: A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32uC in 7% CO2. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSD1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSD1423/1422 and subsequent passage in broth restored CLC expression. LVSD1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD50, respectively. Following immunization, mice challenged IN with over 700 times the LD50 of LVS remained healthy and asymptomatic. Conclusions: Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.

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