Assessing Patient and Provider Perceptions of Factors Associated with Patient Engagement in Asthma Care.

RATIONALE: National quality improvement initiatives emphasize building partnerships between patients and providers by promoting patient engagement through communication, shared decision-making, and self-care skills. Efforts to promote patient engagement are especially important for people with asthma. To cultivate effective partnerships in asthma care, patients and providers may benefit from understanding each other's values and perceptions regarding treatment goals, shared decision-making, as well as barriers to optimal care and outcomes. OBJECTIVES: We conducted a survey study to assess and compare asthma patient and provider perceptions of factors that are associated with effective partnerships and patient engagement. METHODS: Surveys were administered to adult patients with poorly controlled asthma (n = 328) and their physicians (n = 40) before they participated in collaborative learning sessions held in 40 allergy and immunology practices across the United States. The surveys included items for both groups to report their asthma-related treatment goals and perceptions about information needs and knowledge, shared decision-making, and barriers to medication adherence. RESULTS: Providers rated their knowledge about different aspects of their patients' health status (on a scale from 1 = poor knowledge to 5 = excellent knowledge). The lowest percentages of ratings 4 and 5 were for knowledge about patients' financial status (29%), adherence (42%), lifestyle (46%), and workplace situation (46%). The highest percentages of ratings 4 and 5 were for knowledge about patients' exacerbation history (75%), smoking status (76%), hospitalization history (79%), and comorbidities (79%). The percentages of patients and providers, respectively, who indicated the following treatment goals as important differed significantly: preventing exacerbations (62% and 83%; P = 0.01), preventing emergency department visits (44% and 76%; P < 0.01), and improving ability to perform daily activities (69% and 48%; P < 0.01). However, there were no significant differences in percentages of provider-reported goals and goals that providers estimated their patients would indicate as important. Disconnects were also observed for perceived barriers to asthma medication adherence. CONCLUSIONS: The observed disconnects in patient and provider perceptions may inform strategies for cultivating effective partnerships and patient engagement to improve care quality and outcomes for people with asthma.

Rapid degradation of the complement regulator, CD59, by a novel inhibitor.

There is increased interest in immune-based monoclonal antibody therapies for different malignancies because of their potential specificity and limited toxicity. The activity of some therapeutic monoclonal antibodies is partially dependent on complement-dependent cytolysis (CDC), in which the immune system surveys for invading pathogens, infected cells, and malignant cells and facilitates their destruction. CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that protects cells from CDC. However, in certain tumors, CD59 expression is enhanced, posing a significant obstacle for treatment, by hindering effective monoclonal antibody-induced CDC. In this study, we used non-small lung carcinoma cells to characterize the mechanism of a novel CD59 inhibitor: the 114-amino acid recombinant form of the 4th domain of intermedilysin (rILYd4), a pore forming toxin secreted by Streptococcus intermedius. We compared the rates of internalization of CD59 in the presence of rILYd4 or anti-CD59 antibodies and determined that rILYd4 induces more rapid CD59 uptake at early time points. Most significantly, upon binding to rILYd4, CD59 is internalized and undergoes massive degradation in lysosomes within minutes. The remaining rILYd4·CD59 complexes recycle to the PM and are shed from the cell. In comparison, upon internalization of CD59 via anti-CD59 antibody binding, the antibody·CD59 complex is recycled via early and recycling endosomes, mostly avoiding degradation. Our study supports a novel role for rILYd4 in promoting internalization and rapid degradation of the complement inhibitor CD59, and highlights the potential for improving CDC-based immunotherapy.

Scratching the surface: actin' and other roles for the C-terminal Eps15 homology domain protein, EHD2.

The C-terminal Eps15 homology domain-containing (EHD) proteins participate in multiple aspects of endocytic membrane trafficking. Of the four mammalian EHD proteins, EHD2 appears to be the most disparate, both in terms of sequence homology, and in subcellular localization/function. Since its initial description as a plasma membrane-associated protein, the precise function of EHD2 has remained enigmatic. Various reports have suggested roles for EHD2 at the plasma membrane, within the endocytic transport system, and even in the nucleus. For example, EHD2 facilitates membrane fusion/repair in muscle cells. Recently the focus has shifted to the role of EHD2 in regulating caveolae. Indeed, EHD2 is highly expressed in tissues rich in caveolae, including fat, muscle and blood vessels. This review highlights cumulative evidence linking EHD2 to actin-rich structures at the plasma membrane, where the plasma membrane-associated phospholipid phosphatidylinositol 4,5-bisphosphate controls EHD2 recruitment. Herein we examine the key pathways where EHD2 might function, and address its potential involvement in these processes.

Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.

The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

Impact of beta 2-microglobulin on tapasin expression and covalent association.

Cellular immunity is dependent on major histocompatibility complex (MHC) class I molecules enabling cytotoxic T cell recognition of malignant and infected cells. Loading of antigenic peptides onto MHC class I is assisted by a peptide-loading protein complex including tapasin. We found that tapasin expression is enhanced by beta 2-microglobulin via both transcriptional and post-transcriptional mechanisms. In addition, using conditions which preserve the tapasin-ERp57 disulfide-bonded conjugate, we demonstrated that beta 2-microglobulin increases tapasin-containing protein complexes, and reduces the level of MHC class I/ERp57 complexes lacking tapasin. Overall, our results provide a new perspective on the regulation of tapasin expression and association.

Productive association between MHC class I and tapasin requires the tapasin transmembrane/cytosolic region and the tapasin C-terminal Ig-like domain.

The current model of antigen assembly with major histocompatibility complex (MHC) class I molecules posits that interactions between the tapasin N-terminal immunoglobulin (Ig)-like domain and the MHC class I peptide-binding groove permit tapasin to regulate antigen selection. Much less is known regarding interactions that might involve the tapasin C-terminal Ig-like domain. Additionally, the tapasin transmembrane/cytoplasmic region enables tapasin to bridge the MHC class I molecule to the transporter associated with antigen processing (TAP). In this investigation, we made use of two tapasin mutants to determine the relative contribution of the tapasin C-terminal Ig-like domain and the tapasin transmembrane/cytoplasmic region to the assembly of MHC class I molecules. Deletion of a loop within the tapasin C-terminal Ig-like domain (Δ334-342) prevented tapasin association with the MHC class I molecule K(d). Although tapasin Δ334-342 did not increase the efficiency of K(d) folding, K(d) surface expression was enhanced on cells expressing this mutant relative to tapasin-deficient cells. In contrast to tapasin Δ334-342, a soluble tapasin mutant lacking the transmembrane/cytoplasmic region retained the ability to bind to K(d) molecules, but did not facilitate K(d) surface expression. Furthermore, when soluble tapasin and tapasin Δ334-342 were co-expressed, soluble tapasin had a dominant negative effect on the folding and surface expression of not only K(d), but also D(b) and K(b). In addition, our molecular modeling of the MHC class I-tapasin interface revealed novel potential interactions involving tapasin residues 334-342. Together, these findings demonstrate that the tapasin C-terminal and transmembrane/cytoplasmic regions are critical to tapasin's capacity to associate effectively with the MHC class I molecule.

Effect of a tapasin mutant on the assembly of the mouse MHC class I molecule H2-K(d).

Major histocompatibility complex (MHC) class I heavy chain/beta(2)m heterodimers assemble with antigenic peptides through interactions with peptide-loading complex proteins, including tapasin and ERp57. In human cells, a cysteine residue within tapasin (C95) has been shown to form a covalent bond with ERp57. In this study, we focused on the effect of this tapasin amino-acid residue in mouse cells expressing the MHC class I molecule H2-K(d). We showed that a large disulfide-bonded complex was present in the mouse cells that included ERp57, tapasin, and K(d). Furthermore, in mouse cells, unlike human cells, we found that tapasin mutated at C95 can participate in a non-covalent complex with ERp57. Comparison of our findings to earlier findings with a human molecule (HLA-B(*)4402) also revealed that a tapasin C95 mutation has a stronger effect on the maturation and stability of K(d) than HLA-B(*)4402. Overall, our results characterize the influence of this tapasin cysteine residue on the stable surface expression of a mouse MHC class I molecule and reveal differences in tapasin C95 interactions and effects between mouse and human systems.

A transmembrane tail: interaction of tapasin with TAP and the MHC class I molecule.

The transmembrane protein tapasin has an essential role in the assembly of stable major histocompatibility (MHC) class I/peptide complexes. Within the endoplasmic reticulum, tapasin associates with both the transporter associated with antigen processing (TAP) and the MHC class I molecule. The tapasin/TAP association has been clearly shown to involve the transmembrane domains (TMDs) of both molecules and to result in the stable expression of TAP. Although the influence of tapasin on MHC class I molecule folding and surface expression has been extensively studied, relatively little is known at the structural level regarding the interaction between tapasin and the MHC class I molecule. Here we summarize our current understanding of functions involving the tapasin TMD and propose that, beyond stabilizing TAP, the tapasin TMD may also interact with the MHC class I heavy chain.

Amyloid precursor-like protein 2 association with HLA class I molecules.

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2K(d) and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression.

Influence of the tapasin C terminus on the assembly of MHC class I allotypes.

Several endoplasmic reticulum proteins, including tapasin, play an important role in major histocompatibility complex (MHC) class I assembly. In this study, we assessed the influence of the tapasin cytoplasmic tail on three mouse MHC class I allotypes (H2-K(b), -K(d), and -L(d)) and demonstrated that the expression of truncated mouse tapasin in mouse cells resulted in very low K(b), K(d), and L(d) surface expression. The surface expression of K(d) also could not be rescued by human soluble tapasin, suggesting that the surface expression phenotype of the mouse MHC class I molecules in the presence of soluble tapasin was not due to mouse/human differences in tapasin. Notably, soluble mouse tapasin was able to partially rescue HLA-B8 surface expression on human 721.220 cells. Thus, the cytoplasmic tail of tapasin (either mouse or human) has a stronger impact on the surface expression of murine MHC class I molecules on mouse cells than on the expression of HLA-B8 on human cells. A K408W mutation in the mouse tapasin transmembrane/cytoplasmic domain disrupted K(d) folding and release from tapasin, but not interaction with transporter associated with antigen processing (TAP), indicating that the mechanism whereby the tapasin transmembrane/cytoplasmic domain facilitates MHC class I assembly is not limited to TAP stabilization. Our findings indicate that the C terminus of mouse tapasin plays a vital role in enabling murine MHC class I molecules to be expressed at the surface of mouse cells.

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines.

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.