Methods comparison of two-dimensional gel electrophoresis for host cell protein characterization.

Two-dimensional electrophoresis (2DE) is a gel-based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS-PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two-dimensional difference gel electrophoresis (2D-DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non-traditional method of 2D-DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP-based applications.

Microcirculatory and glycocalyx properties are lowered by high-salt diet but augmented by Western diet in genetically heterogeneous mice.

Many individuals in industrialized societies consume a high-salt, Western diet(WD); however, the effects of this diet on microcirculatory properties and glycocalyx barrier function are unknown. Young genetically heterogeneous male and female mice underwent 12 wk of normal chow (NC) diet, NC diet with 4% salt (NC4%), Western diet (WD), or WD with 4% salt (WD4%). Microcirculatory properties and glycocalyx barrier function were evaluated in the mesenteric microcirculation, using an intravital microscope equipped with an automated capture and analysis system. Total microvascular density summed across 4- to 25-µm microvessel segment diameters was lower in NC4% than in NC and WD (P < 0.05). Perfused boundary region (PBR), a marker of glycocalyx barrier function, averaged across 4- to 25-µm microvessel segment diameters was similar between NC and NC4%, as well as between WD and WD4% (P > 0.05). PBR was lower in WD and WD4% than in NC and NC4% (P < 0.05), indicating augmented glycocalyx barrier function in WD and WD4%. There were strong, inverse relationships between PBR and adiposity and blood glucose (r = -0.44 to -0.61, P < 0.05). In summary, NC4% induces deleterious effects on microvascular density, whereas WD augments glycocalyx barrier function. Interestingly, the combination of high-salt, Western diet in WD4% resulted in lower total microvascular density like NC4% and augmented glycocalyx barrier function like WD. These data suggest distinct microcirculatory adaptations to high-salt and Western diets that coincide when these diets are combined in young genetically heterogeneous male and female mice.NEW & NOTEWORTHY Many individuals in industrialized societies consume a combination of high-salt and Western diet; however, the effects of this diet on microcirculatory and glycocalyx properties are unknown. This study reveals that a high-salt diet lowers microcirculatory and glycocalyx properties, whereas a Western diet augments glycocalyx barrier function and thickness. Taken together, these data indicate that there are distinct microcirculatory adaptations to high-salt and Western diets that coincide when high-salt and Western diets are combined.

Occupational Stigma Communication: The Anticipatory Socialization of Sex Educators.

Controversies about sex education have complex, yet often overlooked, occupational implications related to stigma for teachers. In this study, we interviewed 26 future sex educators in their last year of certification about how their anticipatory socialization experiences spoke to the management of potential occupational stigma. Our analysis revealed two stigma management communication (SMC) strategies future sex educators learned, strategies we term cooperation and opportunism, and identified the ways in which those strategies were responses to stigma content cues of responsibility and peril, respectively. We contend that the interactivity of stigma communication is an important site for the theorizing of as-yet-unidentified SMC strategies, strategies that can be enlisted in a diversity of health education and healthcare contexts.

The authoritative metaphor and social change: Surgeon General C. Everett Koop's Direct Mailer, "Understanding AIDS".

In 1988, Surgeon General C. Everett Koop published "Understanding AIDS," the nation's first and only direct mailing sent to every private home in the country. His appeals therein were driven by what we label authoritative metaphors. Communicated by and/or attributed to persons of authority, authoritative metaphors capitalize on the symbolic force of sanctioned power by appealing to the ethos of office. In "Understanding AIDS," we find that Koop drew from his positions as a surgeon and a general, respectively, to equate AIDS with an unprecedented plague and an unprecedented war. He created new authoritative metaphors out of the vestiges of familiar metaphors related to disease and public health and thereby portrayed AIDS as a recognizable but decisively unique dilemma requiring distinct preventative behaviors.

Combined heat and power systems: economic and policy barriers to growth.

BACKGROUND: Combined Heat and Power (CHP) systems can provide a range of benefits to users with regards to efficiency, reliability, costs and environmental impact. Furthermore, increasing the amount of electricity generated by CHP systems in the United States has been identified as having significant potential for impressive economic and environmental outcomes on a national scale. Given the benefits from increasing the adoption of CHP technologies, there is value in improving our understanding of how desired increases in CHP adoption can be best achieved. These obstacles are currently understood to stem from regulatory as well as economic and technological barriers. In our research, we answer the following questions: Given the current policy and economic environment facing the CHP industry, what changes need to take place in this space in order for CHP systems to be competitive in the energy market? METHODS: We focus our analysis primarily on Combined Heat and Power Systems that use natural gas turbines. Our analysis takes a two-pronged approach. We first conduct a statistical analysis of the impact of state policies on increases in electricity generated from CHP system. Second, we conduct a Cost-Benefit analysis to determine in which circumstances funding incentives are necessary to make CHP technologies cost-competitive. RESULTS: Our policy analysis shows that regulatory improvements do not explain the growth in adoption of CHP technologies but hold the potential to encourage increases in electricity generated from CHP system in small-scale applications. Our Cost-Benefit analysis shows that CHP systems are only cost competitive in large-scale applications and that funding incentives would be necessary to make CHP technology cost-competitive in small-scale applications. CONCLUSION: From the synthesis of these analyses we conclude that because large-scale applications of natural gas turbines are already cost-competitive, policy initiatives aimed at a CHP market dominated primarily by large-scale (and therefore already cost-competitive) systems have not been effectively directed. Our recommendation is that for CHP technologies using natural gas turbines, policy focuses should be on increasing CHP growth in small-scale systems. This result can be best achieved through redirection of state and federal incentives, research and development, adoption of smart grid technology, and outreach and education.

Allergen-specific CD8(+) T cells and atopic disease.

Considerable evidence suggests that IL-10 may have a role in the manifestation of atopic disease. We sought to test the hypothesis that at the single cell level, allergen-specific T cells have diminished IL-10 production capacity in severely affected atopics compared with asymptomatic atopics. We defined three A*0201-restricted Der p 1 CD8(+) T cell epitopes. Using human leukocyte antigen-A*0201-peptide (HLA-A*0201-peptide) tetrameric complexes and enzyme-linked immunospot assays to analyze peripheral blood mononuclear cells from A*0201-positive severely symptomatic atopics, asymptomatic atopics, and nonatopic controls, we observed a significant association between the frequency of the Der p 1-specific CD8(+) T cells and disease activity. The specific T cells expressed an antigen-experienced cell surface phenotype, and 45.7% were positive for cutaneous lymphocyte-associated antigen. The specific T cells were able to produce IFN-gamma efficiently, but their IL-10 production was significantly reduced in severely affected atopics. In contrast, viral-specific CD8(+) T cells were able to produce equivalent amounts of IL-10 in the severely affected atopics compared with asymptomatic atopics and nonatopics. Through defining the first human atopic allergen HLA class I epitopes, we have provided a possible cellular mechanism to link the previous association of low IL-10 levels and severe atopic disease. These data are consistent with a role for CD8(+) T cells in atopic disease pathogenesis and may provide a basis for future T cell immunotherapy strategies.

Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection.

Deletion, anergy, and a spectrum of functional impairments can affect virus-specific CD8 cells in chronic viral infections. Here we characterize a low frequency population of CD8 cells present in chronic hepatitis B virus (HBV) infection which survive in the face of a high quantity of viral antigen. Although they do not appear to exert immunological pressure in vivo, these CD8 cells are not classically "tolerant" since they proliferate, lyse, and produce antiviral cytokines in vitro. They are characterized by altered HLA/peptide tetramer reactivity, which is not explained by TCR down-regulation or reduced functional avidity and which can be reversed with repetitive stimulation. CD8 cells with altered tetramer binding appear to have a specificity restricted to envelope antigen and not to other HBV antigens, suggesting that mechanisms of CD8 cell dysfunction are differentially regulated according to the antigenic form and presentation of individual viral antigens.

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections.

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

Dynamics of T cell responses in HIV infection.

Cytotoxic CD8(+) T cells play a major role in the immune response against viruses. However, the dynamics of CD8(+) T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8(+) T cells, in relation to Ag-specific CD4(+) T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8(+) T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.