ABSTRACT
Topical prevention of HIV and other STIs is a global health priority. To provide options for users, developers have worked to design safe, effective and acceptable vaginal dissolving film formulations. We aimed to characterize user experiences of vaginal film size, texture and color, and their role in product-elicited sensory perceptions (i.e. perceptibility), acceptability and willingness to use. In the context of a user-centered product evaluation study, we elicited users' 'first impressions' of various vaginal film formulation designs via visual and tactile prototype inspection during a qualitative user evaluation interview. Twenty-four women evaluated prototypes. Participants considered size and texture to be important for easy insertion. Color was more important following dissolution than prior to insertion. When asked to combine and balance all properties to arrive at an ideal film, previously stated priorities for individual characteristics sometimes shifted, with the salience of some individual characteristics lessening when multiple characteristics were weighted in combination. While first impressions alone may not drive product uptake, users' willingness to initially try a product is likely impacted by such impressions. Developers should consider potential users' experiences and preferences in vaginal film design. This user-focused approach is useful for characterizing user sensory perceptions and experiences relevant to early design of prevention technologies.
Subject(s)
Anti-HIV Agents/administration & dosage , Anti-HIV Agents/chemistry , HIV Infections/prevention & control , Vaginal Creams, Foams, and Jellies/administration & dosage , Vaginal Creams, Foams, and Jellies/chemistry , Administration, Intravaginal , Anti-Infective Agents, Local/administration & dosage , Anti-Infective Agents, Local/chemistry , Chemistry, Pharmaceutical/methods , Female , Humans , Male , Sexually Transmitted Diseases/prevention & controlABSTRACT
Prior to binding to antigenic peptide, the major histocompatibility complex (MHC) heavy chain associates with an assembly complex of proteins that includes calreticulin, tapasin, and the transporter associated with antigen processing (TAP). Our data show that calreticulin can bind weakly to Ld without tapasin's assistance, and that deglycosylation of the alpha1 domain results in a primary loss of binding to calreticulin rather than tapasin. We have also shown that high amounts of wild-type tapasin are still unable to associate with MHC class I in the absence of the MHC class I/calreticulin interaction, confirming the central role of calreticulin in the formation of the MHC class I assembly complex.
Subject(s)
Antiporters/physiology , Calcium-Binding Proteins/metabolism , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Immunoglobulins/physiology , Ribonucleoproteins/metabolism , ATP-Binding Cassette Transporters/metabolism , Antibodies, Monoclonal/immunology , Antigen Presentation , Calreticulin , Cells, Cultured , Humans , Membrane Transport Proteins , Protein Binding , Protein Structure, TertiaryABSTRACT
Prior to the binding of peptide in the endoplasmic reticulum (ER), the major histocompatibility complex (MHC) class I heavy chain associates with an assembly complex that includes the transporter associated with antigen processing (TAP). The proximity of a part of the MHC class I alpha2 domain alpha-helix to areas previously shown to influence assembly complex binding suggests that this region might also be involved in chaperone association. Position 151, found in this part of the alpha2 domain alpha-helix, has a side chain that points up, away from direct contact with peptide, and is occupied by a glycine in all murine MHC class I heavy chains. We found that substitution of this glycine in H-2L(d) with a histidine substantially increased the proportion of peptide-free forms, although TAP binding was not abrogated. Thus, interaction of the heavy chain with peptides, but not with the assembly complex, is influenced by this glycine.
Subject(s)
H-2 Antigens/chemistry , H-2 Antigens/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigen Presentation , Binding Sites , Cell Line , Glycine/chemistry , Histocompatibility Antigen H-2D , Macromolecular Substances , Mice , Models, Molecular , Molecular Chaperones/metabolism , Mutagenesis, Site-Directed , Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-L(d) cells. One mutant has a deletion of nine amino acid residues (tapasin Delta334-342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with L(d) and their effects on L(d) surface expression. We found that tapasin Delta334-342 was unable to bind to the L(d) H chain, and yet it facilitated L(d) assembly and expression. Tapasin Delta334-342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of L(d). Tapasin mutant H334F/H335Y, unlike tapasin Delta334-342, bound to L(d). Expression of tapasin H334F/H335Y in 721.220-L(d) reduced the proportion of cell surface open forms of L(d) and retarded the migration of L(d) from the endoplasmic reticulum. In total, our results indicate that the 334-342 region of tapasin influences L(d) assembly and transport.
