Parental communication satisfaction with the clinical team in the paediatric cardiac ICU.

INTRODUCTION: Understanding parents' communication preferences and how parental and child characteristics impact satisfaction with communication is vital to mitigate communication challenges in the cardiac ICU. METHODS: This cross-sectional survey was conducted from January 2019 to March 2020 in a paediatric cardiac ICU with parents of patients admitted for at least two weeks. Family satisfaction with communication with the medical team was measured using the Communication Assessment Tool for Team settings. Clinical characteristics were collected via Epic, Pediatric Cardiac Critical Care Consortium local entry and Society for Thoracic Surgeons Congenital Heart Surgery Databases. Associations between communication score and parental mood, stress, perceptions of clinical care, and demographic characteristics along with patient demographic and clinical characteristics were examined. Multivariable ordinal models were conducted with characteristics significant in bivariate analysis. RESULTS: In total, 93 parents of 84 patients (86% of approached) completed surveys. Parents were 63% female and 70% White. Seventy per cent of patients were <6 months old at admission, 25% had an extracardiac abnormality, and 80% had a cardiac surgery this admission. Parents of children with higher pre-surgical risk of mortality scores (OR 2.875; 95%CI 1.076-7.678), presence of surgical complications (72 [63.0, 75.0] vs. 64 [95%CI 54.6, 73] (p = 0.0247)), and greater satisfaction with care in the ICU (r = 0.93922; p < 0.0001) had significantly higher communication scores. CONCLUSION: These findings can prepare providers for scenarios with higher risk for communication challenges and demonstrate the need for further investigation into interventions that reduce parental anxiety and improve communication for patients with unexpected clinical trajectories.

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina.

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)-based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Leber's hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.

Oritavancin Retains a High Affinity for a Vancomycin-Resistant Cell-Wall Precursor via Its Bivalent Motifs of Interaction.

Despite its potent antibacterial activities against drug-resistant Gram-positive pathogens, oritavancin remains partially understood with respect to its primary mode of hydrogen bond interaction with a cell-wall peptide regarding the role of its lipophilic 4'-chlorobiphenyl moiety. Here we report a surface plasmon resonance (SPR) study performed in two cell-wall model surfaces, each prepared by immobilization with a vancomycin-susceptible Lys-d-Ala-d-Ala or vancomycin-resistant Lys-d-Ala-d-Lac peptide. Analysis of binding kinetics performed on the peptide surface showed that oritavancin bound ∼100-1000-fold more tightly than vancomycin on each model surface. Ligand competition experiments conducted by SPR and fluorescence spectroscopy provided evidence that such affinity enhancement can be attributed to its 4'-chlorobiphenyl moiety, possibly through a hydrophobic interaction that led to a gain of free energy with a contribution from enthalpy as suggested by a variable-temperature SPR experiment. On the basis of these findings, we propose a model for the bivalent motifs of interaction of oritavancin with cell-wall peptides, by which the drug molecule can retain a strong interaction even with the vancomycin-resistant peptide. In summary, this study advances our understanding of oritavancin and offers new insight into the significance of bivalent motifs in the design of glycopeptide antibiotics.

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

rAAV-compatible MiniPromoters for restricted expression in the brain and eye.

BACKGROUND: Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. METHODS: For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. RESULTS: The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. CONCLUSIONS: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

Urinary cytokines/chemokines pattern in patients with painful bone metastases undergoing external beam radiotherapy experiencing pain flare.

BACKGROUND: External beam radiotherapy (EBRT) is a mainstay for treatment of painful bone metastases. Transient worsening of pain ("pain flare") occurs in 40% of patients. We investigated the pathophysiology of pain flare through assessment of changes in urinary cytokines/chemokines in patients receiving EBRT for painful bone metastases. METHODS: Urine samples were collected from patients receiving a single 8 Gy fraction for painful bone metastases preparation, day 1 or 2 and on an additional day between days 3 to 5 post radiation. Patients completed a standardized pain and analgesic use diary daily for 10 days following radiation. Patients were deemed to have pain flare if they had a two-point increase from baseline worst pain on 0-10 scale and no decrease in analgesic intake or a 25% increase in analgesic intake with no decrease in worst pain. The Millipore Milliplex 42-Plex Cyto-kine/Chemokine Kit™ was used to measure urinary levels of a panel of cytokines/chemokines. RESULTS: Forty-six patients consented to the study of which 28 were evaluable (complete urine and diary data), and 83/84 urine samples were available for analysis. Pain flare was experienced by 11 patients (39%). The following cytokines/chemokines were detectable in at least 50% of the patients: EGF, fractalkine, GRO, IL-4, IL-8, interferon gamma induced protein 10 (IP-10), MCP-1, macrophage derived chemokine (MDC), PDGF-AA, sIL-2Ra, TGF-Alpha, VEGF. Comparing patients with or without pain flare EGF, fractalkine, GRO, IL-8, IP-10, MCP-1, MDC, sIL-2Ra, and TGF-alpha increased following radiation in both groups. Patients with pain flare have significant lower levels on IL-8, IP-10, and MDC over time. No specific time trend was noticed. CONCLUSIONS: Patients who experience pain flare appear to have a different pattern in urinary cytokine/chemokine levels than patients without pain flare. A larger study is required to confirm the possible role of cytokines/chemokines in predisposition to and/or the cause of pain flare following radiation to painful bone metastases.

Targeted CNS Delivery Using Human MiniPromoters and Demonstrated Compatibility with Adeno-Associated Viral Vectors.

Critical for human gene therapy is the availability of small promoter tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters using computational biology and phylogenetic conservation. MiniPromoters were tested in mouse as single-copy knock-ins at the Hprt locus on the X Chromosome, and evaluated for lacZ reporter expression in CNS and non-CNS tissue. Eighteen novel MiniPromoters driving expression in mouse brain were identified, two MiniPromoters for driving pan-neuronal expression, and 17 MiniPromoters for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPromoters exhibit similar cell-type specificity when delivered via adeno-associated virus (AAV) vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy number effects or genomic location, and results in constructs predisposed to success in AAV. These MiniPromoters are immediately applicable for pre-clinical studies towards gene therapy in humans, and are publicly available to facilitate basic and clinical research, and human gene therapy.

Adverse events across generations of bone-modifying agents in patients with solid tumor cancers reported in Phase III randomized trials.

AIMS: The objective of this study is to compare adverse events experienced among different bone-modifying agents. METHODS: A literature search was conducted to identify Phase III bisphosphonate and bone-modifying agent trials reporting adverse effects. Thirty-seven adverse events of interest were identified for six different treatment options. Weighted linear regression modeling was performed on the adverse event proportions with treatment groups, normalized through applying natural log transformations. RESULTS: There were significant differences in adverse events of vomiting (p = 0.045) and osteonecrosis of the jaw (p = 0.017), and combined item events of nausea/vomiting (p = 0.048), hematological and lymphatic system toxicities (p = 0.020), and any respiratory system problem (p = 0.023) between bone-modifying agent and placebo trials. The significant toxicities were observed even after adjusting for the two confounding factors of age and primary cancer site. CONCLUSION: While adverse effects are consistently experienced more frequently in patients receiving bone-modifying agents when compared with placebos, we find that the majority of individual side effects are not significantly more frequent in patients receiving bone-modifying agents compared with placebo.

Symptom clusters in patients with metastatic cancer: a literature review.

This article reviews the literature reporting empirically determined symptom clusters in patients with metastatic cancer. A literature search was conducted on symptom clusters within heterogeneous metastatic cancer patient populations using MEDLINE, EMBASE, and CINAHL. Studies examining predetermined symptom clusters were excluded. A total of eight relevant studies published between 2005 and 2011 were identified. The number of symptom clusters extracted varied from two to eight clusters per study, comprising of two to eight symptoms per cluster. There were no clusters consistently identified within all eight studies. Notable differences in symptoms assessed, assessment tools, statistical analysis, patient demographics were observed between the studies. The lack of consensus among the inter-study symptom clusters are likely due to the differences in patient population as well as study methodology. Further exploration in metastatic symptom cluster research will ideally improve patient outcomes by facilitating improved symptom management in future clinical practice.

New Considerations in the Design of Clinical Trials for Bone Metastases.

Palliative radiotherapy (RT) is prescribed to patients with bone metastases to alleviate symptoms and improve quality of life. The lack of consistent endpoints for such trials has made cross study comparison difficult and has led to contradictory conclusions. The International Bone Metastases Consensus Working Party was established to create a standard set of endpoints and recommendations for future clinical trials. Recommendations were included regarding eligibility criteria, pain assessments, follow-up assessments, timing, as well as radiation techniques. Suggestions were also made to facilitate the ease with which different studies could be compared as well as to encourage widespread consistency in certain aspects of trial design. Investigators conducting clinical trials in bone metastases should continue to adopt these recommendations to ensure consistent guidelines based on the most recent literature.

The utilization of telephone follow-up in the advanced cancer population: a review of the literature.

BACKGROUND: Palliative cancer patients often require clinic or hospital follow-up after any treatment intervention they may have received. This is typically done in person at either a hospital or a clinic. In these advanced cancer patients, this may be burdensome and result in attrition. Telephone follow-up is becoming more frequently used as an adjunct to clinical follow-up. It can be conducted for both clinical trials, as well as interventional purposes. The purpose of this study was to review the literature and examine the utility and effectiveness of telephone follow-up in the advanced cancer population. METHODS: A literature search was conducted on Medline (1980 - April week 4 2012), Embase (1980 - week 17 2012), the Cochrane Central Register of Controlled Trials (April 2012) and CINAHL (1981-July 31 2012). RESULTS: A total of 11 studies were identified that were published between 2001 and 2011. All studies were in the clinical trial setting. Studies that utilized telephone follow-up in the advanced cancer population, as well as studies that compared the feasibility of telephone follow-up with hospital follow-up, were included in this review. Follow-up at week 4 (month 1) was the most common interval for patient contact. Information collected during the contact varied with the study; however, the most commonly used tool was the Edmonton Symptom Assessment System. Other information included analgesic diary, patient feedback, satisfaction with the care and post-treatment side effects, along with a variety of quality of life questionnaires. Some studies provided information to the patient about protocols for care, advice and coping strategies. Attrition was common even with the use of telephone contact in place of clinical follow-up. CONCLUSION: Telephone follow-up is a feasible alternative to traditional hospital follow-ups for assessment of symptom palliation. There are fewer burdens on the patient, allowing for a better maintenance of quality of life and lower rates of attrition in clinical trials. Patients had an overall positive opinion of the use of this alternative approach with no common disadvantages. A combination of follow-up strategies, such as clinic follow-up and telephone contact for those not attending, may result in a more comprehensive assessment.