Improving chronic disease care by adding laypersons to the primary care team: a parallel randomized trial.

BACKGROUND: Improving the quality and efficiency of chronic disease care is an important goal. OBJECTIVE: To test whether patients with chronic disease working with lay "care guides" would achieve more evidence-based goals than those receiving usual care. DESIGN: Parallel-group randomized trial, stratified by clinic and conducted from July 2010 to April 2012. Patients were assigned in a 2:1 ratio to a care guide or usual care. Patients, providers, and persons assessing outcomes were not blinded to treatment assignment. (ClinicalTrials.gov: NCT01156974). SETTING: 6 primary care clinics in Minnesota. PATIENTS: Adults with hypertension, diabetes, or heart failure. INTERVENTION: 2135 patients were given disease-specific information about standard care goals and asked to work toward goals for 1 year, with or without the help of a care guide. Care guides were 12 laypersons who received brief training about these diseases and behavior change. MEASUREMENTS: The primary end point for each patient was change in percentage of goals met 1 year after enrollment. RESULTS: The percentage of goals met increased in both the care guide and usual care groups (changes from baseline, 10.0% and 3.9%, respectively). Patients with care guides achieved more goals than usual care patients (82.6% vs. 79.1%; odds ratio, 1.31 [95% CI, 1.16 to 1.47]; P < 0.001); reduced unmet goals by 30.1% compared with 12.6% for usual care patients; and improved more than usual care patients in meeting several individual goals, including not using tobacco. Estimated cost was $286 per patient per year. LIMITATIONS: Providers' usual care may have been influenced by contact with care guides. Last available data in the electronic health record were used to assess end points. CONCLUSION: Adding care guides to the primary care team can improve care for some patients with chronic disease at low cost.

Care guides: an examination of occupational conflict and role relationships in primary care.

BACKGROUND: Improving the efficiency and effectiveness of primary care treatment of patients with chronic illness is an important goal in reforming the U.S. health care system. Reducing occupational conflicts and creating interdependent primary care teams is crucial for the effective functioning of new models being developed to reorganize chronic care. Occupational conflict, role interdependence, and resistance to change in a proof-of-concept pilot test of one such model that uses a new kind of employee in the primary care office, a "care guide," were analyzed. Care guides are lay individuals who help chronic disease patients and their providers achieve standard health goals. PURPOSE: The aim of this study was to examine the development of occupational boundaries, interdependence of care guides and primary care team members, and acceptance by clinic employees of this new kind of health worker. METHODOLOGY/APPROACH: A mixed methods, pilot study was conducted using qualitative analysis; clinic, provider, and patient surveys; administrative data; and multivariate analysis. Qualitative analysis examined the emergence of the care guide role. Administrative data and surveys were used to examine patterns of interdependence between care guides, physicians, team members, and clinic staff; obtain physician evaluations of the care guide role; and evaluate the effect of care guides on patient perceptions of care coordination and follow-up. FINDINGS: Evaluation of implementation of the care guide model showed that (a) the care guide scope of practice was clearly defined; (b) interdependent relationships between care guides and providers were formed; (c) relational triads consisting of patient, care guide, and physician were created; (d) patients and providers were supported in managing chronic disease; and (e) resistance to this model among traditional employees was minimized. PRACTICE IMPLICATIONS: The feasibility of implementing a new care model for chronic disease management in the primary care setting, identifying factors associated with a positive organizational experience, was shown in this study.

Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.

The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

Human cytomegalovirus temporally regulated gene expression in differentiated, immortalized retinal pigment epithelial cells.

BACKGROUND: Human cytomegalovirus (HCMV) replication in epithelial cells is crucial for its pathogenesis. To date, HCMV gene expression has been primarily studied in human foreskin fibroblasts (HFFs), although their importance for HCMV pathogenesis remains unclear. Primary retinal pigment epithelial (RPE) cells are permissive for HCMV. OBJECTIVES: Our objectives were to determine the production of alternatively processed HCMV major immediate-early (MIE) and UL37 RNAs and their essential products in infected, terminally differentiated immortalized RPE (hTERT-RPE) cells. STUDY DESIGN: hTERT-RPE cells were studied because of their notable similarities with primary RPE cells, and because they overcome key limitations of primary cells. hTERT-RPE cells were terminally differentiated in vitro and infected with HCMV. Total RNA or cell proteins were analyzed at various times post-infection. RESULTS: We show for the first time that HCMV-infected, differentiated hTERT-RPE cells produce IE1, IE2, UL37 exon 1 (UL37x1) and UL37 alternatively spliced RNAs, albeit with abundances and kinetics distinct from those observed in HCMV-infected HFFs. IE1-72 was produced in HCMV-infected, differentiated hTERT-RPEs within 24h post-infection (hpi); whereas, IE2-86 and pUL37x1 were produced within 72 hpi. IE2-86 was detected after IE1-72 even though its transcript appeared first. Early/late (pp65) and late (pp28) proteins were produced within 96-120 hpi. CONCLUSIONS: The temporal cascade of HCMV gene expression was observed in infected, differentiated hTERT-RPE cells. Moreover, HCMV IE RNAs are alternatively and accurately processed in differentiated hTERT-RPE cells. However, the delayed temporal expression suggests further regulation of HCMV gene expression at post-transcriptional/translational levels in differentiated hTERT-RPE cells.

Access to health care: differences between insured and uninsured patients in south Minneapolis.

This article reports the results of a survey of 575 patients who were seen at 2 clinics in south Minneapolis. About half had health insurance. We found a strong connection between not having insurance and going without medical care. We also found that when the uninsured do go for care, they often fail to take prescribed medications because of the cost. Our findings also contradicted some prevalent thinking about the uninsured: that they are unemployed, that they refuse employer-sponsored insurance, and that they can easily get care at free clinics.

Do drug samples influence resident prescribing behavior? A randomized trial.

PURPOSE: The purpose of the study was to determine whether access to drug samples influences resident prescribing decisions. SUBJECTS AND METHODS: The authors observed 390 decisions to initiate drug therapy by 29 internal medicine residents over a 6-month period in an inner-city primary care clinic. By random selection, half of the residents agreed not to use available free drug samples. Five drug class pairs were chosen for study prospectively. Highly advertised drugs were matched with drugs commonly used for the same indication that were less expensive, available over-the-counter, or available in generic formulation. RESULTS: Resident physicians with access to drug samples were less likely to choose unadvertised drugs (131/202 decisions) than residents who did not have access to samples (138/188 decisions; P = .04) and less likely to choose over-the-counter drugs (51/202, 73/188; P = .003). There was a trend toward less use of inexpensive drugs. CONCLUSION: Access to drug samples in clinic influences resident prescribing decisions. This could affect resident education and increase drug costs for patients.

Posting guidelines: a practical and effective way to promote appropriate hypertension treatment.

BACKGROUND: Despite publication and periodic updating of treatment guidelines, hypertension remains undertreated in the United States, and physicians underuse recommended drugs. METHODS: Hypertension treatment guidelines were summarized and posted in five places in a hospital-based primary care clinic staffed by internists and internal medicine residents. Costs and recommended doses of five commonly used antihypertensive drugs were included. The charts of all 253 patients seen during a four-month period with a diagnosis of hypertension were analyzed. Blood pressures and physician prescribing habits were compared at baseline and at 8, 12, and 16 months after posting the guidelines. RESULTS: The number of patients with blood pressures < 140/90 mm Hg increased from 41% to 58%, p = .001. Median (IQR) systolic pressure fell from 143 (119-167) to 137 (116-158) mm Hg, p < .0001 and diastolic pressure from 78 (65-91) to 77 (64-90) mm Hg, p = .0002. Physicians prescribed more recommended drugs, more total antihypertensive drugs, larger doses of hydrochlorothiazide and lisinopril, and more inexpensive drugs. The total cost of antihypertensive drugs per patient increased slightly. CONCLUSION: Regular exposure to clinical guidelines, presented in a practical and simple way, can change physician behavior and improve patient care.

Care guides: employing nonclinical laypersons to help primary care teams manage chronic disease.

Lay persons ("care guides") without previous clinical experience were hired by a primary care clinic, trained for 2 weeks, and assigned to help 332 patients and their providers manage their diabetes, hypertension, and congestive heart failure. One year later, failure by these patients to meet nationally recommended guidelines was reduced by 28%, P < .001. Improvement was seen in tobacco usage, blood pressure control, pneumonia vaccination, low-density lipoprotein cholesterol levels, annual eye examinations, aspirin use, and microalbuminuria testing. Care guides served an average of 111 patients at an annual per patient cost of $392. Further testing of this model is warranted.

Specific interaction of hepatitis C virus glycoproteins with mannan binding lectin inhibits virus entry.

Mannan-binding lectin (MBL) is a soluble innate immune protein that binds to glycosylated targets. MBL acts as an opsonin and activates complement, contributing to the destruction and clearance of infecting microorganisms. Hepatitis C virus (HCV) encodes two envelope glycoproteins E1 and E2, expressed as non-covalent E1/E2 heterodimers in the viral envelope. E1 and E2 are potential ligands for MBL. Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment, the full-length E1/E2 heterodimer, expressed in vitro, and assess the effect of this interaction on virus entry. A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent, saturating binding of MBL to HCV glycoproteins. Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL. MBL binds to E1/E2 representing a broad range of virus genotypes. MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles (HCVpp) bearing E1/E2 from a wide range of genotypes. HCVpp were neutralized to varying degrees. MBL was also shown to neutralize an authentic cell culture infectious virus, strain JFH-1 (HCVcc). Furthermore, binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2. In conclusion, MBL interacts directly with HCV glycoproteins, which are present on the surface of the virion, resulting in neutralization of HCV particles.

Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA.

A characteristic of many positive-strand RNA viruses is that, whilst replication of the viral genome is dependent on the expression of the majority of non-structural proteins in cis, virus particle formation can occur when most or all of the structural proteins are co-expressed in trans. Making use of a recently identified hepatitis C virus (HCV) isolate (JFH1) that can be propagated in tissue culture, this study sought to establish whether this is also the case for hepaciviruses. Stable cell lines containing one of two bicistronic replicons derived from the JFH1 isolate were generated that expressed non-structural proteins NS3-5B or NS2-5B. Release and transmission of these replicons to naïve Huh7 cells could then be demonstrated when baculovirus transduction was used to express the HCV proteins absent from the subgenomic replicons. Transmission could be blocked by a neutralizing antibody targeted at the E2 envelope protein, consistent with this phenomenon occurring via trans-encapsidation of replicon RNA into virus-like particles. Transmission was also dependent on expression of NS2, which was most effective at promoting virus particle formation when expressed in cis on the replicon RNA compared with in trans via baculovirus delivery. Density gradient analysis of the particles revealed the presence of a broad infectious peak between 1.06 and 1.11 g ml(-1), comparable to that seen when propagating full-length virus in tissue culture. In summary, the trans-encapsidation system described offers a complementary and safer approach to study HCV particle formation and transmission in tissue culture.

Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein.

The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1-6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.

Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32.

The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

Diabetes control improved when inner-city patients received graphic feedback about glycosylated hemoglobin levels.

OBJECTIVE: To develop and test an inexpensive visual tool to help patients with diabetes improve glycemic control. METHODS: A multidisciplinary team developed a 1-page form, the "Take-home Diabetes Record" (THDR), providing feedback to patients by displaying per cent glycosylated hemoglobin (GHb) values graphically over time, with target levels highlighted. Patients with type 2 diabetes in an inner-city clinic were randomized to THDR use (n = 57) or not (n = 70) over 15 months. Self-care activities were discussed, linked with GHb results, and charted at each clinic visit. Initial and final GHb were compared. RESULTS: Mean GHb fell significantly in THDR patients (-0.94, P =.003), but not in control patients (-0.18, P =.36). Mean GHb decrease was greater in THDR patients (P =.047). A greater proportion of THDR patients (51%) than control patients (18%) achieved a decrease in GHb >/=0.9 (P =.001). CONCLUSIONS: A graph linking GHb and self-care activities shows promise for improving glycemic control.

Complex alternative processing of human cytomegalovirus UL37 pre-mRNA.

Differentially processed human cytomegalovirus (HCMV) UL37 RNAs encode biologically significant proteins. Due to the recent discovery of alternative UL37 exon 3 (UL37x3) splice donors, permissively infected cells were thoroughly examined for additional alternatively spliced UL37 RNAs. Newly described donors within UL37 exon 1 (nt 52520) and intron 1 (nt 52209) as well as UL37x3 di (nt 50770) and dii (nt 50782) were differentially spliced to known downstream UL37 acceptors. The alternatively spliced UL37(S), UL37(L), UL37(di) and UL37d(ii) RNAs predictably encode proteins of 83, 163, 217 and 213 residues, respectively, which share UL37x1 N-terminal sequences but differ downstream in their C termini. Moreover, temporal expression of the alternatively spliced UL37 RNAs differs during HCMV infection. The complexity of UL37 pre-mRNA processing is evidenced by the detection of 11 UL37 spliced and unspliced UL37x1 RNAs in HCMV-infected cells. Based upon these data, a revised HCMV UL37 gene map is presented, which incorporates all RNA species detected during permissive infection.

Convergence of RNA cis elements and cellular polyadenylation factors in the regulation of human cytomegalovirus UL37 exon 1 unspliced RNA production.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for its growth. The UL37 promoter drives production of an unspliced and several alternatively spliced RNAs. The UL37 exon 1 (UL37x1) unspliced RNA is abundant from IE to late times of HCMV infection, whereas the UL37 spliced RNAs are markedly less abundant. Production of the UL37x1 unspliced RNA requires polyadenylation (PA) at nucleotide 50998, which lies within intron 1, upstream of the UL37 exon 2 (UL37x2) acceptor. The physical proximity of its cis elements suggests steric hindrance between PA and splicing machineries for UL37 pre-mRNA. To test this possibility, we generated site-specific mutants in Target 1 PA and RNA splicing cis elements and compared the PA and splicing efficiencies of mutant RNAs with those of wild-type RNA. The mutually exclusive processing events of UL37x1 PA and UL37x1-UL37x2 splicing have been accurately recapitulated in transfected permissive human fibroblasts (HFFs) expressing a Target 1 minigene RNA, which contains the required splicing and PA cis elements. Two mutants in the invariant PA signal dramatically decreased UL37x1 PA as expected and, concomitantly, increased the efficiency of UL37x1-UL37x2 RNA splicing. Consistent with these results, changes to consensus UL37x1 donor and UL37x2 acceptor sites increased the efficiency of UL37x1-UL37x2 RNA splicing but decreased the efficiency of UL37x1 PA. Moreover, HCMV infection of HFFs increased the abundance of the PA cleavage stimulatory factor CstF-64, the potent splicing suppressor PTB, and the hypophosphorylated form of the splicing factor SF2 at 4 h postinfection. Induction of these factors further favors production of the UL37x1 unspliced RNA over that of the spliced RNAs. Taken together, these results suggest that there is a convergence in UL37 RNA regulation by cis elements and cellular proteins which favors production of the UL37x1 unspliced RNA during HCMV infection at the posttranscriptional level.

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection.

Alternative processing of human cytomegalovirus (HCMV) UL37 pre-mRNA predominantly produces the unspliced UL37 exon 1 (UL37x1) RNA and multiple, lower abundance, alternatively spliced UL37 RNAs. The relative abundance of UL37x1 unspliced RNA is surprising because it requires the favoured use of a polyadenylation signal within UL37 intron 1, just upstream of the UL37 exon 2 (UL37x2) acceptor. Here, it was shown that a downstream element (DSE) in UL37x2 strongly enhanced processing at the UL37x1 polyadenylation site, but did not influence UL37x1-x2 splicing. There was a potential binding site (UCUU) for polypyrimidine tract-binding protein (PTB) at the UL37x1 polyadenylation/cleavage site and its mutation to UGGG reduced both polyadenylation and splicing of UL37x1-x2 minigene pre-mRNA, suggesting a role in both RNA processing events. To determine whether lytic HCMV infection altered the balance of RNA processing factors, which bind to UL37 pre-mRNA cis elements, these were investigated in permissively infected primary and immortalized human diploid fibroblasts (HFFs) and epithelial cells. Induction of polyadenylation factors in HCMV-infected, serum-starved (G(0)) HFFs was also investigated. Permissive HCMV infection consistently increased, albeit with different kinetics, the abundance of cleavage stimulation factor 64 (CstF-64) and PTB, and altered hypo-phosphorylated SF2 in different cell types. Moreover, the preponderance of UL37x1 RNA increased during infection and correlated with CstF-64 induction, whereas the complexity of the lower abundance UL37 spliced RNAs transiently increased following reduction of hypo-phosphorylated SF2. Collectively, multiple UL37 RNA polyadenylation cis elements and induced cellular factors in HCMV-infected cells strongly favoured the production of UL37x1 unspliced RNA.

Human cytomegalovirus UL37 immediate early target minigene RNAs are accurately spliced and polyadenylated.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt -82 to -143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3'-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

The products of human cytomegalovirus genes UL23, UL24, UL43 and US22 are tegument components.

We have investigated the human cytomegalovirus (HCMV) US22 gene family members UL23, UL24, UL43 and US22. Specific antibodies were generated to identify pUL23 (33 kDa), pUL24 (40 kDa) and pUL43 (48 kDa), while pUS22 was identified by monoclonal antibody HWLF1. A C-terminally truncated UL43 product (pUL43t; 21 kDa) produced by a deletion mutant was also investigated. The UL24 and UL43 genes were expressed with early-late (gamma1) and true-late (gamma2) kinetics, respectively. Immunoblot and immuno-EM studies demonstrated that pUL23, pUL24, pUL43 and pUS22 were virion tegument components. Immunofluorescence and immuno-EM studies showed that pUL23, pUL24, pUL43 and pUL43t were located in cytoplasmic protein aggregates, manifesting two forms: complex juxtanuclear structures and smaller, membrane-bound aggregates resembling dense bodies. The complex-type aggregate is a putative site of particle maturation. Because pUL43t was present in protein aggregates, but under-represented in virus particles compared to pUL43, it was concluded that N-terminal sequences target pUL43 to protein aggregates and that C-terminal sequences are important for incorporation into particles. Since three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components, at least seven of the twelve US22 family genes encode tegument proteins, suggesting that the products of the remaining five genes might be similarly located. These findings demonstrate a common biological feature among most, if not all, US22 family proteins and implicate the family in events occurring immediately after virus penetration.