Biomedical subjects

Kristina K Peachman

Publications and source records attributed to Kristina K Peachman.

8 recordsLinked to original sources

PubMed2007

Restricted replication and lysosomal trafficking of yellow fever 17D vaccine virus in human dendritic cells.

The yellow fever virus attenuated 17D vaccine strain is a safe and effective vaccine and a valuable model system for evaluating immune responses against attenuated viral variants. This study compared the in vitro interactions of the commercially available yellow fever vaccine (YF-VAX), Dengue virus and the live-attenuated dengue vaccine PDK50 with dendritic cells (DCs), the main antigen-presenting cells at the initiation of immune responses. Similar to PDK50, infection with YF-VAX generated activated DCs; however, for YF-VAX, activation occurred with limited intracellular virus replication. The majority of internalized virus co-localized with endolysosomal markers within 90 min, suggesting that YF-VAX is processed rapidly in DCs. These results indicate that restricted virus replication and lysosomal compartmentalization may be important contributing factors to the success of the YF-VAX vaccine.

Animals↗

PubMed2006

Multicomponent anthrax toxin display and delivery using bacteriophage T4.

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to Hoc can be displayed on the same capsid and such particles can elicit broad immunological responses. Anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their functional domains, were fused to Hoc with an N-terminal hexa-histidine tag and the recombinant proteins were over-expressed in E. coli and purified. Using a defined in vitro assembly system, the anthrax-Hoc fusion proteins were efficiently displayed on T4 capsid, either individually or in combinations. All of the 155 Hoc binding sites can be occupied by one antigen, or they can be split among two or more antigens by varying their molar ratio in the binding reaction. Immunization of mice with T4 phage carrying PA, LF, and EF elicited strong antigen-specific antibodies against all antigens as well as lethal toxin neutralization titers. The triple antigen T4 phage elicited stronger PA-specific immune responses than the phage displaying PA alone. These features offer novel avenues to develop customized multicomponent vaccines against anthrax and other pathogenic diseases.

Animals↗

PubMed2006

Correlation between lethal toxin-neutralizing antibody titers and protection from intranasal challenge with Bacillus anthracis Ames strain spores in mice after transcutaneous immunization with recombinant anthrax protective antigen.

Transcutaneous immunization of mice with recombinant protective antigen (rPA) of Bacillus anthracis resulted in significantly higher lethal toxin-neutralizing antibody titers than did intramuscular injection of alum-adsorbed rPA. Immunized mice were partially protected against intranasal challenge with 235,000 (10 50% lethal doses) Ames strain B. anthracis spores. A highly significant correlation was observed between toxin-neutralizing antibody titer and survival after challenge. Future experiments with rabbits and nonhuman primates should confirm the significance of protection by this vaccine strategy.

Administration, Cutaneous↗

PubMed2005

Human dendritic cells and macrophages exhibit different intracellular processing pathways for soluble and liposome-encapsulated antigens.

The intracellular fates of soluble and liposomal antigens in human macrophages and dendritic cells are not well defined. Previous studies using murine macrophages have demonstrated that liposomal antigens can enter the MHC class I pathway. The Golgi complex is a major organelle in this pathway. Phagocytosis of the antigens is followed by translocation of antigen-derived peptides to the trans-Golgi where they can complex with MHC class I molecules. In contrast, soluble antigens are normally processed through the MHC class II pathway. Therefore, in the present study, ovalbumin and a synthetic Ebola peptide were used either in a soluble form or encapsulated in liposomes to investigate the intracellular trafficking and localization of these antigens to the Golgi complex in human macrophages and dendritic cells. While liposome-encapsulated antigens were transported to the trans-Golgi region in 59-78% of macrophages, soluble antigens remained diffuse throughout the cytoplasm with only 3-11% of the macrophages exhibiting trans-Golgi localization. The majority of dendritic cells localized both soluble (Ebola, 75%; ovalbumin, 84%) and liposomal antigens (58% and 65%), and irradiated Ebola virus to the trans-Golgi. These studies demonstrate that the intracellular fate of soluble and liposomal antigens can differ depending upon the antigen-presenting cell.

Biological Transport↗

PubMed2004

Functional microtubules are required for antigen processing by macrophages and dendritic cells.

Antigen-presenting cells readily phagocytose antigens and channel them through various membrane-bound organelles within the cell. In previous studies, we demonstrated that macrophages concentrated and localized particulate antigens to the trans-Golgi prior to displaying the MHC-class I-antigenic peptides on the cell surface. In this study, we evaluated the importance of cytoskeletal elements in the intracellular trafficking of soluble and liposome-encapsulated ovalbumin in murine bone marrow-derived macrophages and human dendritic cells. F-actin, as identified by staining with fluorescein phalloidin, was observed at the point of contact between soluble or liposomal antigen and the cell membrane, suggesting that a rearrangement of the cytoskeleton occurs to facilitate the uptake of the antigens. Cells were incubated with colchicine, a microtubule depolymerizing agent, or paclitaxel, a microtubule polymerizing agent, before the addition of Texas Red-labeled ovalbumin or liposome-encapsulated Texas Red-labeled ovalbumin. Colchicine disrupted the trans-Golgi, whereas the trans-Golgi complexes were intact in paclitaxel treated cells. In either paclitaxel or colchicine-treated macrophages, internalized liposomal ovalbumin was not concentrated in the area of the trans-Golgi as determined by staining with fluorescent ceramide. In contrast, soluble ovalbumin was concentrated in the region of the trans-Golgi in 15% of the dendritic cells treated with paclitaxel, whereas 6% of the dendritic cells were able to concentrate liposomal antigen. In colchicine-treated dendritic cells, both soluble and liposomal antigens were internalized but did not localize to the area of the trans-Golgi. These data suggest that trafficking of soluble and liposome-encapsulated ovalbumin requires a functional microtubule-dependent translocation system.

Animals↗

PubMed2004

Modulation of dengue virus infection of dendritic cells by Aedes aegypti saliva.

Dengue virus (DV) is a flavivirus carried by the Aedes aegypti mosquito that causes a spectrum of illnesses in the tropics, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dendritic cells (DCs) are professional antigen presenting cells recently shown to be permissive for DV, and implicated as the primary targets of initial DV infection. DV is transmitted to human host by infected mosquitoes during a blood meal, but it is currently unknown whether transmission is modified by vector saliva that is also deposited in the host's skin during feeding. Previous studies evaluated only the outcome of DV infection of DCs, and did not address the influence of mosquito saliva. To more fully characterize natural transmission of DV, we evaluated the effects of Ae. aegypti saliva on DV infection of human myeloid DCs. We found that saliva inhibited DV infection in DCs. Moreover, pre-sensitization of DCs with saliva, prior to DV infection, enhanced inhibition. In addition, enhanced production of IL-12p70 and TNF-alpha were detected in DV-infected DC cultures exposed to mosquito saliva. The proportion of dead cells was also significantly reduced in these cultures. These data contribute to the overall understanding of the natural pathogenesis of DV infection and suggest that there is a protective role for mosquito saliva that limits viral uptake by DCs.

Aedes↗

PubMed2003

Immunization with DNA through the skin.

The skin has evolved as a barrier to prevent external agents, including pathogens, from entering the body. It has a complex and efficient immune surveillance system, which includes Langerhans cells and dendritic cells. By targeting the body's natural defense system, skin-DNA immunization attempts to produce an efficient immune response. Nucleic acid vaccines provide DNA for protein expression in a variety of cells, including keratinocytes, Langerhans cells, and dendritic cells, which are located in the two main areas of the skin, the epidermis (the most superficial layer) and the dermis. After maturation, Langerhans cells and dermal dendritic cells can migrate to local lymph nodes where presentation of antigens to T cells can occur and thus start a variety of immunologic responses. Dermal immunization methods described in this article target the epidermis, the dermis, or both and include: (a) stripping; (b) chemical modification; (c) trans-epidermal immunization (transcutaneous immunization or non-invasive vaccination of the skin); (d) gene gun technology; (e) electroporation; (f) intradermal injections; and (g) microseeding. These techniques all require the removal of hair, the circumvention or modification of the stratum corneum layer of the epidermis, and the addition of DNA or amplification of DNA signal. As the biology of the skin and the mechanisms of DNA vaccination are elucidated, these skin immunization techniques will be optimized. With refinement, skin-DNA immunization will achieve the goal of producing a reliable and efficacious immune response to a variety of pathogens.

Administration, Cutaneous↗

PubMed2003

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.

Animals↗