Redesigning care to support earlier discharge from a neonatal intensive care unit: a design thinking informed pilot.

BACKGROUND: Preterm infants may remain in neonatal intensive care units (NICUs) to receive proper nutrition via nasogastric tube feedings. However, prolonged NICU stays can have negative effects for the patient, the family and the health system. AIM: To demonstrate how a patient-centred, design thinking informed approach supported the development of a pilot programme to enable earlier discharge of preterm babies. METHOD: We report on our design thinking-empathy building approach to programme design, initial outcomes and considerations for ongoing study. RESULTS: Through the use of design thinking methods, we identified unique needs, preferences and concerns that guided the development of our novel early discharge programme. We found that stable, preterm infants unable to feed by mouth and requiring nasogastric tubes can be cared for at home with remote patient monitoring and telehealth support. In addition, novel feeding strategies can help address parental preferences without compromising infant growth. CONCLUSION: A patient-centred, design thinking informed approach supported the development of a pilot programme to enable earlier discharge of preterm babies. The programme resulted in a reduced length of stay, thereby increasing NICU bed capacity and limiting hospital turn-aways.

Preparing for the future: multi-stakeholder perspectives on autonomous goal setting for adolescents with autism spectrum disorders.

PURPOSE: Self-determination is linked to numerous positive outcomes including improved social inclusion and quality of life for adolescents with autism spectrum disorder. Autonomous participation in therapeutic goal setting is an important component of self-determination. This study examined perceptions of, and barriers to, autonomous goal setting as perceived by: (1) adolescents with autism spectrum disorder, (2) parents of adolescents with autism spectrum disorder, and (3) interdisciplinary professionals. METHOD: Three focus groups (one with each of the key stakeholders) and one joint parent and child interview were conducted. All discussions were audio-recorded, transcribed verbatim and thematically analyzed based on the methods outlined by Braun and Clarke (2006). RESULTS: A total of 7 parents (6 mothers, 1 father), 4 youth with autism spectrum disorder, and 10 professionals participated in the study. Three themes related to current experiences with goal setting emerged: (1) purpose, (2) barriers, and (3) types. CONCLUSIONS: Currently, adolescents with autism spectrum disorder are rarely active participants in the goal setting process. This study provides novel data, from the perspectives of multiple stakeholders, about barriers, perceptions, and actualities related to autonomous goal setting for adolescents with autism spectrum disorder. Implications for rehabilitation Adolescent participation in autonomous goal setting is an important component of self-determination. Rehabilitation professionals, parents, and adolescents with autism spectrum disorder identified similar barriers to autonomous goal setting. Adolescents with autism spectrum disorder described goals as short-term, concrete tasks, whereas parents perceived involvement in goal setting as a gateway to long-term self-determination. Rehabilitation professionals and parents acknowledged the benefits and advocated for increased involvement of adolescents with autism spectrum disorder in the goal setting process. Similar to the paradigm shift toward family-centered care, rehabilitation professionals need to shift to include the involvement and participation of individuals with autism spectrum disorder.

Obesity prevalence and related risk of comorbidities among HIV+ patients attending a New England ambulatory centre.

BACKGROUND: Human immunodeficiency virus infection and obesity are pro-inflammatory conditions that, when occurring together, may pose a synergistic risk for diabetes and cardiovascular disease. PURPOSE: The aim of the current study was (i) to document the prevalence of obesity in HIV+ patients treated at the Miriam Hospital Immunology Center (Providence, RI) and (ii) to investigate the relationship between obesity and comorbidities. METHODS: The study population consisted of 1,489 HIV+ adults (70% men; average age 48 ± 11 years) treated between 01/01/2012 and 06/30/2014. Separate logistic regressions tested the associations between overweight and obesity and comorbid diagnoses (diabetes, hypertension and cardiovascular disease), as compared with normal weight. Covariates included age, gender and smoking status. RESULTS: Approximately 37% of patients were overweight (body mass index 25.0-29.9), and an additional 28% were obese (body mass index ≥30.0). Obesity was associated with higher odds of comorbid diabetes (OR = 3.26, CI = 1.98-5.39) and hypertension (OR = 2.11, CI = 1.49-2.98). There was no significant association between obesity and the presence of cardiovascular disease (OR = 1.12, CI = 0.66-1.90). Overweight was associated only with higher odds of comorbid diabetes (OR = 1.72; CI = 1.02-2.88). CONCLUSION: Our findings demonstrate a heightened risk of comorbidities in overweight and obese HIV + patients. Future studies should investigate whether weight loss interventions for this population can reduce cardiovascular and metabolic risk factors as they do in other populations.

The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes.

High-risk human papillomaviruses (HPV) are the etiological pathogen of cervical and a number of ano-genital cancers. How HPVs overcome the significant barriers of the skin immune system has been the topic of intensive research. The E6 and E7 oncoproteins have emerged as key players in the deregulation of host innate immune pathways that are required for the recruitment of effector cells of the immune response. Here we demonstrate that E7, and to a lesser extend E6, strongly reduce NFκB activation in response to the inflammatory mediator imiquimod. Moreover, we establish that undifferentiated keratinocytes do not express the putative receptor for imiquimod, TLR7, and as such are stimulated by imiquimod through a novel pathway. Inhibition of imiquimod induced cytokine production required residues in the CR1 and CR3 regions of E7 and resulted in reduced nuclear translocation and acetylation of the p65 sub-unit of NFκB. The results provide further evidence for a TLR7-independent role of imiquimod in the epithelial immune response and reinforce the ability of the HPV oncoproteins to disrupt the innate immune response, which may have important consequences for establishment of a chronic infection.

Human papillomavirus E7 oncoprotein increases production of the anti-inflammatory interleukin-18 binding protein in keratinocytes.

UNLABELLED: Human papillomavirus (HPV) can successfully evade the host immune response to establish a persistent infection. We show here that expression of the E7 oncoprotein in primary human keratinocytes results in increased production of interleukin-18 (IL-18) binding protein (IL-18BP). This anti-inflammatory cytokine binding protein is a natural antagonist of IL-18 and is necessary for skin homeostasis. We map increased IL-18BP production to the CR3 region of E7 and demonstrate that this ability is shared among E7 proteins from different HPV types. Furthermore, mutagenesis shows that increased IL-18BP production is mediated by a gamma-activated sequence (GAS) in the IL-18BP promoter. Importantly, the increased IL-18BP levels seen in E7-expressing keratinocytes are capable of diminishing IL-18-mediated CD4 lymphocyte activation. This study provides the first evidence for a virus protein that targets IL-18BP and further validates E7 as a key component of the HPV immune evasion armor. IMPORTANCE: Infection with human papillomavirus is a leading cause of morbidity and mortality worldwide. This study demonstrates that the E7 protein increases production of the anti-inflammatory IL-18BP, a major regulator of epithelial homeostasis. A number of E7 proteins can increase IL-18BP production, and a region within the CR3 of E7 is necessary for mediating the increase. A consequence of increased IL-18BP production is a reduction in CD4-positive lymphocyte activation in response to IL-18 costimulation. These findings may shed light on the immune evasion abilities of HPV.

Merkel cell polyomavirus small T antigen targets the NEMO adaptor protein to disrupt inflammatory signaling.

Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKß-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aß, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell.

Human immunodeficiency viruses: propagation, quantification, and storage.

Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long-term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting.

Putting the brakes on the anti-viral response: negative regulators of type I interferon (IFN) production.

Type I IFNs (IFNα/ß) are essential anti-viral cytokines produced in response to the detection of viral components by host pattern recognition receptors. IFNα/ß production is transient, and aberrant activation can be hazardous to the host. In this article, we review our current understanding of host negative regulatory mechanisms that control IFNα/ß production.

Modulation of HIV-1 macrophage-tropism among R5 envelopes occurs before detection of neutralizing antibodies.

HIV-1 R5 viruses vary widely in their capacity to infect primary macrophages. R5 macrophage-tropism is associated with an increased envelope:CD4 affinity that partly results from an increased exposure of CD4 contact residues on gp120 and allows the use of low levels of CD4 for infection. The selective pressures in vivo that modulate R5 macrophage-tropism are not understood. It is possible that different R5 variants adapt for replication in either T-cells (high CD4) or in macrophages (low CD4). However, other selective pressures in vivo (e.g. neutralizing antibodies) may also impact R5 tropism. Here, we measured macrophage infectivity conferred by gp120 sequences amplified sequentially from subjects in London followed from the acute stage of infection. We report wide variation in the capacity of these envelopes to confer macrophage infection in the complete absence of both autologous and heterologous neutralizing antibodies. Our data show that the variation in macrophage tropism observed at early times cannot have been influenced by neutralizing antibodies.

Optineurin negatively regulates the induction of IFNbeta in response to RNA virus infection.

The innate immune response provides a critical defense against microbial infections, including viruses. These are recognised by pattern recognition receptors including Toll-like receptors (TLRs) and RIG-I like helicases (RLHs). Detection of virus triggers signalling cascades that induce transcription of type I interferons including IFNbeta, which are pivotal for the initiation of an anti-viral state. Despite the essential role of IFNbeta in the anti-viral response, there is an incomplete understanding of the negative regulation of IFNbeta induction. Here we provide evidence that expression of the Nemo-related protein, optineurin (NRP/FIP2), has a role in the inhibition of virus-triggered IFNbeta induction. Over-expression of optineurin inhibited Sendai-virus (SeV) and dsRNA triggered induction of IFNbeta, whereas depletion of optineurin with siRNA promoted virus-induced IFNbeta production and decreased RNA virus replication. Immunoprecipitation and immunofluorescence studies identified optineurin in a protein complex containing the antiviral protein kinase TBK1 and the ubiquitin ligase TRAF3. Furthermore, mutagenesis studies determined that binding of ubiquitin was essential for both the correct sub-cellular localisation and the inhibitory function of optineurin. This work identifies optineurin as a critical regulator of antiviral signalling and potential target for future antiviral therapy.

Expression of the NS3 protease of cytopathogenic bovine viral diarrhea virus results in the induction of apoptosis but does not block activation of the beta interferon promoter.

Bovine viral diarrhea virus (BVDV; genus Pestivirus) can exist as two biotypes, cytopathogenic (CP) and non-cytopathogenic (NCP). The CP form differs from NCP by the continual expression of free non-structural protein 3 (NS3). CP BVDV infection of cultured cells induces apoptosis, whereas NCP BVDV infection has been reported to block the induction of beta interferon (IFN-beta). To investigate the viral mechanisms underlying these effects, NS3 or NS2-3 proteins of NCP and CP BVDV biotypes, together with the cognate NS3 co-factor NS4A, were expressed in cells, and their effect on apoptosis and induction of IFN-beta was investigated. Expression of NS3/4A resulted in increased activity of caspase-9 and caspase-3, indicating induction of the intrinsic apoptosis pathway. Mutational analysis revealed that a protease-inactive NS3/4A was unable to induce apoptosis, suggesting that NS3 protease activity is required for initiation of apoptosis during CP BVDV infection. The ability of NS2-3 to modulate activation of the IFN-beta promoter was also investigated. These studies confirmed that, unlike the related hepatitis C virus and GB virus-B, BVDV proteases are unable to inhibit TLR3- and RIG-I-dependent activation of the IFN-beta promoter. These data suggest that BVDV NS3/4A is responsible for regulating the levels of cellular apoptosis and provide new insights regarding the viral elements associated with CP biotype pathogenesis.

Continuous positive airway pressure therapy reduces right ventricular volume in patients with obstructive sleep apnea: a cardiovascular magnetic resonance study.

STUDY OBJECTIVES: There are few data on the effects of continuous positive airway pressure (CPAP) therapy on the structural and functional characteristics of the right heart in patients with obstructive sleep apnea (OSA). We sought to leverage the advantages of cardiac magnetic resonance imaging (CMR) and hypothesized that CPAP treatment would improve right ventricular (RV) function in a group of patients with OSA who were free of other comorbid conditions. METHODS: Patients with severe (apnea-hypopnea index > or = 30/h) untreated OSAwere prospectively enrolled. CMR included 3-dimensional measurement of biventricular size and function, and rest/stress myocardial perfusion and was performed at baseline and after 3 months of CPAP therapy. RESULTS: Fifteen patients with mild to moderate desaturation were enrolled; 2 could not undergo CMR due to claustrophobia and obesity. There were significant decreases in the Epworth Sleepiness Scale score (p < 0.0001) and RV end-systolic and RV end-diastolic volumes (p < 0.05) with CPAP. There was a trend toward improvement in RV ejection fraction, but the improvement did not reach statistical significance. Other measures such as left ventricular volumes, left ventricular ejection fraction, myocardial perfusion reserve index, and thickness of the interventricular septum and ventricular free wall did not change significantly. CONCLUSIONS: This preliminary study found that CPAP treatment decreases RV volumes in patients with severe OSA who are otherwise healthy. CMR offers a novel technique to determine the effects of CPAP on ventricular structure and function in patients with OSA. A randomized controlled study is needed to confirm the results of our study.

Novel CaV2.1 clone replicates many properties of Purkinje cell CaV2.1 current.

The P-type calcium current is mediated by a voltage-sensing CaV2.1 alpha subunit in combination with modulatory auxiliary subunits. In Purkinje neurones, this current has distinctively slow inactivation kinetics that may depend on alternative splicing of the alpha subunit and/or association with different CaVbeta subunits. To better understand the molecular components of P-type calcium current, we cloned a CaV2.1 cDNA from total mouse brain. The full-length CaV2.1 isoform that we isolated (GenBank AY714490) contains sequences recently shown to be present in Purkinje neurones. In agreement with previously published work, the alternatively spliced amino acid V421, implicated in slow inactivation, was not encoded in AY714490 and was absent from reverse transcription-polymerase chain reaction products generated from single Purkinje cells. Next, we studied the expression of the four known mouse auxiliary CaVbeta2 isoforms in Purkinje neurones. Confirmation of the presence of CaVbeta2a in Purkinje cells, previously shown by others to slow CaV2.1 kinetics, led us to characterize its influence on current dynamics. We studied currents generated by the clone AY714490 coexpressed in tsA201 cells with four different CaVbeta subunits. In addition to the well-documented slowing of open-state inactivation kinetics, coexpression with the CaVbeta2a subunit also protected CaV2.1 channels from closed-state inactivation and prevented the channel from inactivating during physiological trains of action potential-like stimuli. This strong resistance to inactivation parallels the property of Purkinje neurone P-type currents and is suggestive of a role for CaVbeta2a in modulating the inactivation properties of P-type calcium currents in Purkinje neurones.

Differential expression of Na+/K+-ATPase alpha-subunits in mouse hippocampal interneurones and pyramidal cells.

The sodium pump (Na+/K+-ATPase), maintains intracellular and extracellular concentrations of sodium and potassium by catalysing ATP. Three sodium pump alpha subunits, ATP1A1, ATP1A2 and ATP1A3, are expressed in brain. We compared their role in pyramidal cells and a subset of interneurones in the subiculum. Interneurones were identified by their expression of GFP under the GAD-65 promoter. We used the sensitivity to the cardiac glycoside, ouabain, to discriminate between different alpha subunit isoforms. GFP-positive interneurones were depolarized by nanomolar doses of ouabain, but higher concentrations were needed to depolarize pyramidal cells. Comparison of pump currents in these cells revealed a current sensitive to low doses of ouabain in interneurones, while micromolar doses of ouabain were needed to suppress the pump current in subicular pyramidal cells. As predicted, nanomolar doses of ouabain increased the frequency but not the amplitudes of IPSPs in pyramidal cells. Immunostaining confirmed a differential distribution of alpha-subunits of the Na+/K+-ATPase in subicular interneurones and pyramidal cells. In conclusion, these data suggest that while ATP1A3-isoforms regulate sodium and potassium homeostasis in subicular interneurones, ATP1A1-isoforms assume this function in pyramidal cells. This differential expression of sodium pump isoforms may contribute to differences in resting membrane potential of subicular interneurones and pyramidal cells.

Effects of vpu start-codon mutations on human immunodeficiency virus type 1 replication in macrophages.

The human immunodeficiency virus type 1 (HIV-1) vpu protein increases the release of virus particles from infected cells. Mutations that abrogate vpu function have a profound effect on HIV-1 replication in primary macrophage cultures. About 1.24 % of primary isolates in the HIV databases have vpu start-codon mutations. In addition, the envelope of the AD8 isolate was reported to compensate for the lack of vpu, whilst the YU-2 virus (cloned directly from the brain tissue of an infected individual) is macrophage-tropic, despite having a vpu start-codon mutation. These observations raise the possibility that envelopes evolve to compensate for the loss of vpu function in vivo. Chimeric vpu+ and vpu- replication-competent clones were constructed that contained the envelopes of SF162, AD8 or YU-2. Macrophages were infected with these chimeras and virus release was measured over time by a reverse transcriptase ELISA. It was found that vpu-deficient chimeras carrying AD8 and YU-2 envelopes were consistently released at lower levels than their wild-type (wt) vpu counterparts, indicating that these envelopes did not compensate for the lack of vpu. Non-chimeric vpu+ and vpu- AD8 and YU-2 followed similar patterns, although replication by vpu-deficient AD8 was variable, with virion release reaching 60 % of that recorded for AD8 with a wt vpu. In summary, no evidence was found that the AD8 or YU-2 envelopes can compensate for the lack of vpu for replication in macrophages.

The promise of CCR5 antagonists as new therapies for HIV-1.

The chemokine receptors CCR5 and CXCR4 were identified as HIV-1 co-receptors in 1996. Since then, a range of agents that bind these receptors and potently block HIV-1 infection have been described, including monoclonal antibodies, peptides and modified chemokines. However, small organic molecules that bind CCR5 are currently the most promising of the co-receptor antagonists for the potential treatment of HIV. These agents are now in advanced stages of clinical development and should soon augment current therapies, as well as being candidates for inclusion in microbicides. Unlike existing drugs that target HIV proteins (eg, reverse transcriptase and protease), co-receptor antagonists bind receptors encoded by the host. As a consequence, blockade of these receptors may result in immunosuppressive effects or other disorders. Furthermore, co-receptor inhibitors may also be more toxic than currently available HIV therapies, and it is not yet clear whether they will become candidates for first-line therapy. Nonetheless, safer, less toxic versions of such inhibitors may be achievable in the future. The use of CCR5 inhibitors as a second-line treatment increases the possibility that these reagents will select for more pathogenic CXCR4-using variants. The development of effective CXCR4 antagonists for dual treatment would be beneficial; however, whether long-term treatment with antagonists of the widely expressed CXCR4 receptor is feasible without toxicity is unknown. This review discusses the current status of CCR5 antagonists, their modes of action and their development for therapeutic use.

A non-lethal technique for detecting the chytrid fungus Batrachochytrium dendrobatidis on tadpoles.

Batrachochytrium dendrobatidis (Bd) infection on post-metamorphic frogs and salamanders is commonly diagnosed using polymerase chain reaction (PCR) of skin scrapings taken with mildly abrasive swabs. The technique is sensitive, non-lethal, and repeatable for live animals. Tadpoles are generally not sampled by swabbing but are usually killed and their mouthparts excised to test for the pathogen. We evaluated a technique for non-lethal Bd diagnosis using quantitative PCR (qPCR) on swabs scraped over the mouthparts of live tadpoles. The sensitivity of non-lethal (swabbing) and lethal (removal of mouthparts) sampling was assessed using 150 Bd-infected Rana subaquavocalis tadpoles. Swabbing was consistently less sensitive than lethal sampling, but still detected Bd. Experimental Bd prevalence was 41.1% when estimated by destructively sampling mouthparts and 4.7 to 36.6% (mean = 21.4%) when estimated with swabs. Detection rates from swabbing varied with investigator and time since infection. The likelihood of detecting Bd-infected tadpoles was similar regardless of size and developmental stage. Swabbing mouthparts of live tadpoles is a feasible and effective survey technique for Bd, but, because it is less sensitive, more tadpoles must be sampled to estimate prevalence at a confidence level comparable to destructive sampling.

Scaling of diastolic intraventricular pressure gradients is related to filling time duration.

In early diastole, pressure is lower in the apex than in the base of the left ventricle (LV). This early intraventricular pressure difference (IVPD) facilitates LV filling. We assessed how LV diastolic IVPD and intraventricular pressure gradient (IVPG), defined as IVPD divided by length, scale to the heart size and other physiological variables. We studied 10 mice, 10 rats, 5 rabbits, 12 dogs, and 21 humans by echocardiography. Color Doppler M-mode data were postprocessed to reconstruct IVPD and IVPG. Normalized LV filling time was calculated by dividing filling time by RR interval. The relationship between IVPD, IVPG, normalized LV filling time, and LV end-diastolic volume (or mass) as fit to the general scaling equation Y = kM beta, where M is LV heart size parameter, Y is a dependent variable, k is a constant, and beta is the power of the scaling exponent. LV mass varied from 0.049 to 194 g, whereas end-diastolic volume varied from 0.011 to 149 ml. The beta values relating normalized LV filling time with LV mass and end-diastolic volume were 0.091 (SD 0.011) and 0.083 (SD 0.009), respectively (P < 0.0001 vs. 0 for both). The beta values relating IVPD with LV mass and end-diastolic volume were similarly significant at 0.271 (SD 0.039) and 0.243 (SD 0.0361), respectively (P < 0.0001 vs. 0 for both). Finally, beta values relating IVPG with LV mass and end-diastolic volume were -0.118 (SD 0.013) and -0.104 (SD 0.011), respectively (P < 0.0001 vs. 0 for both). As a result, there was an inverse relationship between IVPG and normalized LV filling time (r = -0.65, P < 0.001). We conclude that IVPD decrease, while IVPG increase with decreasing animal size. High IVPG in small mammals may be an adaptive mechanism to short filling times.