Maximizing phenotype constraint and extracellular matrix production in primary human chondrocytes using arginine–glycine–aspartate concentration gradient hydrogels

New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine–glycine–aspartic acid peptide (RGD) concentrations from 0 mM to 10 mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.     

Enhancing Care for Hospitalized Older Adults with Cognitive Impairment: A Randomized Controlled Trial

Background

Approximately 40% of hospitalized older adults have cognitive impairment (CI) and are more prone to hospital-acquired complications. The Institute of Medicine suggests using health information technology to improve the overall safety and quality of the health care system.

Objective

Evaluate the efficacy of a clinical decision support system (CDSS) to improve the quality of care for hospitalized older adults with CI.

Design

A randomized controlled clinical trial.

Setting

A public hospital in Indianapolis.

Population

A total of 998 hospitalized older adults were screened for CI, and 424 patients (225 intervention, 199 control) with CI were enrolled in the trial with a mean age of 74.8, 59% African Americans, and 68% female.

Intervention

A CDSS alerts the physicians of the presence of CI, recommends early referral into a geriatric consult, and suggests discontinuation of the use of Foley catheterization, physical restraints, and anticholinergic drugs.

Measurements

Orders of a geriatric consult and discontinuation orders of Foley catheterization, physical restraints, or anticholinergic drugs.

Results

Using intent-to-treat analyses, there were no differences between the intervention and the control groups in geriatric consult orders (56% vs 49%, P = 0.21); discontinuation orders for Foley catheterization (61.7% vs 64.6%, P = 0.86); physical restraints (4.8% vs 0%, P = 0.86), or anticholinergic drugs (48.9% vs 31.2%, P = 0.11).

Conclusion

A simple screening program for CI followed by a CDSS did not change physician prescribing behaviors or improve the process of care for hospitalized older adults with CI.

Electronic supplementary material

The online version of this article (doi:10.1007/s11606-012-1994-8) contains supplementary material, which is available to authorized users.KEY WORDS: cognitive impairment, clinical trial, decision support, hospitalized elders     

Distance traveled to undergo revision shoulder arthroplasty is impacted by patient demographics and exceeds that of primary shoulder arthroplasty

BackgroundPrevious research has highlighted disparities in access to and outcomes following shoulder arthroplasty. The purpose of this study is to compare travel distance for primary vs. revision surgery and to determine the relationships between travel distance to undergo revision shoulder arthroplasty and patient demographics and postoperative adverse outcomes. We hypothesized that older patients, those with increased medical comorbidities, and those with greater financial resources would travel farther for surgery.MethodsThe Healthcare Cost and Utilization Project, American Hospital Association, and UnitedStatesZipCodes.org Enterprise data sets were compiled to collect data on patient demographics, operation performed, and postoperative adverse outcomes. Population-weighted zip code centroid points of patients’ residences as well as hospital coordinates were used to approximate the distance traveled to undergo shoulder arthroplasty. Kruskal-Wallis tests and logistic regression analyses were used to analyze the relationship between travel distance and patients’ demographics and postoperative outcomes.ResultsPatients traveled farther to undergo revision shoulder arthroplasty than to undergo primary shoulder arthroplasty (P = .0001). Among patients who underwent revision operations, older age (P = .0001) and increased medical comorbidities (P = .003) were associated with decreased travel distance. White patients and those with commercial insurance traveled farther to receive care (P = .0001). There were no clinically significant associations between postoperative outcomes following revision surgery and travel distance.ConclusionPatients may travel farther to undergo revision surgery owing to patient preferences or because these complex operations are performed at fewer centers. Younger and healthier patients, white patients, and those with commercial insurance plans traveled farther to receive care. Future work is needed to determine the underlying causes of these disparities and whether it is patient preferences or systemic factors such as hospital density or access to high-quality health insurance that are influencing access to this specialized surgical care.     

Implementing a screening and diagnosis program for dementia in primary care

BACKGROUND: Primary care physicians are positioned to provide early recognition and treatment of dementia. We evaluated the feasibility and utility of a comprehensive screening and diagnosis program for dementia in primary care. METHODS: We screened individuals aged 65 and older attending 7 urban and racially diverse primary care practices in Indianapolis. Dementia was diagnosed according to International Classification of Diseases (ICD)-10 criteria by an expert panel using the results of neuropsychologic testing and information collected from patients, caregivers, and medical records. RESULTS: Among 3,340 patients screened, 434 scored positive but only 227 would agree to a formal diagnostic assessment. Among those who completed the diagnostic assessment, 47% were diagnosed with dementia, 33% had cognitive impairment—no dementia (CIND), and 20% were considered to have no cognitive deficit. The overall estimated prevalence of dementia was 6.0% (95% confidence interval (CI) 5.5% to 6.6%) and the overall estimate of the program cost was $128 per patient screened for dementia and $3,983 per patient diagnosed with dementia. Only 19% of patients with confirmed dementia diagnosis had documentation of dementia in their medical record. CONCLUSIONS: Dementia is common and undiagnosed in primary care. Screening instruments alone have insufficient specificity to establish a valid diagnosis of dementia when used in a comprehensive screening program; these results may not be generalized to older adults presenting with cognitive complaints. Multiple health system and patient-level factors present barriers to this formal assessment and thus render the current standard of care for dementia diagnosis impractical in primary care settings. Supported by grant R01 HS10884-01 from the Agency for Healthcare Research and Quality.     

Hematologic and immunomodulatory effects of an interleukin-1 receptor antagonist coinfusion during low-dose endotoxemia in healthy humans

Endotoxin is a component of gram-negative bacteria that causes hematologic and immunologic changes through its induction of cytokines. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of IL-1 that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity. We investigated the ability of human recombinant IL-1Ra to block the effects of low-dose endotoxin. Fourteen healthy male volunteers between 18 and 30 years old were injected intravenously with 3 ng/kg Escherichia coli endotoxin. Concurrent with the injections, nine volunteers received a 3-hour continuous intravenous infusion of IL-1Ra. The other five subjects were given a 3-hour infusion of saline. Volunteers injected with endotoxin experienced a threefold increase in circulating neutrophils over baseline. This neutrophilia was significantly reduced by 48% in subjects administered endotoxin plus IL-1Ra (P = .0253). Ex vivo mitogen-induced peripheral blood mononuclear cell proliferation decreased by greater than 60% at 3 and 6 hours after endotoxin injection (P = .0053). This endotoxin-induced reduction in mitogen response was reversed in subjects coinjected with IL-1Ra (P = .0253). Endotoxin-induced symptoms, fever, and tachycardia were unaffected by IL-1Ra. IL-1 appears to be an important mediator in endotoxemia because some of its hematologic and immunomodulatory effects can be blocked by IL-1Ra.     

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.