Evidence-Based Pain Assessment in Nonverbal Palliative Care Patients.

BACKGROUND: Central to palliative care is the early assessment and treatment of pain, whether physical, psychosocial, or spiritual. Nonverbal palliative care patients are at risk for inadequate pain assessment leading to prolonged suffering. AIMS: The purpose of this project was to implement and evaluate an evidence-based pain assessment tool for nonverbal palliative care patients. DESIGN: The Iowa Model Revised: Evidence-Based Practice to Promote Excellence in Healthcare and the Implementation Strategies for Evidence-Based Practice Guide provided the guiding frameworks. SETTINGS: On a six-bed adult inpatient Palliative Care Unit (PCU). PARTICIPANTS/SUBJECTS: Nonverbal palliative care patients. METHODS: Evidence supported use of the Multidimensional Objective Pain Assessment Tool (MOPAT) for nonverbal patients receiving palliative care. During an eight-week pilot, nurses recorded pain assessments on a paper form and trended pain scores over a 24-hour period. Evaluation included knowledge, attitudes, and behaviors pre- and post-pilot and was subsequently used in a Precision Implementation Approach to promote adoption. RESULTS: Nurses' attitudes toward palliative care pain assessment improved in all items on the evaluation tools. Pain was assessed using MOPAT for 74% of nonverbal palliative care patients and 88% of patients had linked pain interventions to MOPAT scores. CONCLUSIONS: MOPAT is the only valid evidence-based pain assessment tool for nonverbal patients receiving palliative care. This project led to successful adoption of the MOPAT within the PCU.

Evaluation of Efficacy, Efficiency, and Cell Viability of a Novel Descemet Membrane Endothelial Keratoplasty Graft Preparation Device, DescePrep, in Nondiabetic and Diabetic Human Donor Corneas.

PURPOSE: The purpose of this study was to evaluate the safety, efficacy, and efficiency of a Descemet membrane endothelial keratoplasty (DMEK) graft preparation device, DescePrep, through measurement of graft viability, yield, and preparation time in both healthy and diabetic (high-risk) donor eyes. METHODS: Twenty nondiabetic and 10 diabetic donor corneas were processed using DescePrep, which standardizes the liquid bubble technique. Corneas were stained with trypan blue and then processed. Cell counts through specular microscopy, optical coherence tomography imaging, and slit-lamp analysis were used for the evaluation of graft separation and viability in 5 nondiabetic corneas. The remaining 25 corneas (15 nondiabetic and 10 diabetic) were evaluated for preparation success rate and processing time. Ten corneas (5 nondiabetic and 5 diabetic) were randomly selected for further evaluation of global cell loss through staining. RESULTS: Ninety-seven percent of corneas (29 of 30) were prepared successfully with DescePrep. The average preparation time was 2.83 ± 1.8 minutes. There was no significant difference in the time of preparation between the nondiabetic and diabetic groups (P = 0.077). The overall average cell death after processing was 7.9% ± 3.7% for all corneas. There was no significant difference in cell viability between diabetic and nondiabetic tissues after DescePrep processing (P = 0.769). CONCLUSIONS: DescePrep is a new DMEK preparation technique that can process both nondiabetic and diabetic donor corneas at high yields in minutes. High-yield preparation of diabetic corneas may offer eye banks access to a larger donor pool, which is important because the demand for DMEK grafts continues to rise worldwide.

Characteristics of benign neuroblastic tumors: Is surgery always necessary?

PURPOSE: Ganglioneuroma (GN) and ganglioneuroblastoma-intermixed (GNB-I) represent benign variants of neuroblastic tumors in children; however, differentiating from more aggressive histological variants of GNB including the nodular subtype (GNB-N) prior to resection can be challenging, even with biopsy. Currently, no standard treatment guidelines exist. The purpose of this study was to identify pre-operative characteristics of benign neuroblastic tumors and evaluate outcomes for patients who underwent surgical resection or observation. METHODS: Retrospective chart review of children treated at a single institution between 2009 and 2019 for non-metastatic tumor with a tissue diagnosis of GN, GNB-N or GNB-I. Demographics, imaging, labs, operative details and outcomes were recorded and analyzed. RESULTS: Of 53 patients, 45% were male. The most common tumor location was abdomen (49%), followed by thorax (34%). Forty-five percent had at least one image defined risk factor. Biopsy was performed in 32% (17/53) and upfront surgery in 68% (36/53). Three patients (3/53, 5.6%) with biopsy demonstrating GN tumors were observed due to high surgical risk. Pathology of resected specimens demonstrated GN in 52% (26/50) and GNB-I or GNB-N in 48% (24/50). The majority of GNB tumors (75% (18/24) were GNB-I and 25% (6/24) were GNB-N. Therefore, 88% of the resected tumors were benign spectrum neuroblastic tumors (GN & GNB-I). Seven (7/50, 14%) patients experienced perioperative complication (temporary paralysis, Horner's syndrome, chylothorax, vocal cord paralysis). Recurrence was noted in 1 patient with GN (1/50, 2%) and 3 with GNB-N (3/50, 6%). There were no tumor-related deaths. Patients with GN were older than those with GNB (8.8 years (IQR 6-11.25) vs 5.6 years for GN (IQR 3-7); p = 0.01). GNB tumors were also more likely to have calcifications on imaging (63% vs. 38%, p = .01) and more commonly had MIBG avidity (88% vs 66%, p = .04). There were no significant differences in tumor size or symptoms at presentation. CONCLUSIONS: In children with neuroblastic tumors, older age, CT without tumor calcifications, lack of MIBG avidity, and/or normal urine catecholamines may indicate benign GN. Close observation could be considered for asymptomatic patients meeting these criteria with biopsy-proven GN, with resection reserved for progressive growth or symptom development. However, larger, multicenter studies are needed for further validation. LEVEL OF EVIDENCE: IV.

Age-associated expression of p21and p53 during human wound healing.

In mice, cellular senescence and senescence-associated secretory phenotype (SASP) positively contribute to cutaneous wound healing. In this proof-of-concept study, we investigated the expressions of p16, p21, and other senescence-associated biomarkers during human wound healing in 24 healthy subjects using a double-biopsy experimental design. The first punch biopsy created the wound and established the baseline. The second biopsy, concentric to the first and taken several days after wounding, was used to probe for expression of biomarkers by immunohistochemistry and RNA FISH. To assess the effects of age, we recruited 12 sex-matched younger (30.2 ± 1.3 years) and 12 sex-matched older (75.6 ± 1.8 years) subjects. We found that p21 and p53, but not p16, were induced during healing in younger, but not older subjects. A role for Notch signaling in p21 expression was inferred from the inducible activation of HES1. Further, other SASP biomarkers such as dipeptidyl peptidase-4 (DPP4) were significantly induced upon wounding in both younger and older groups, whereas matrix metallopeptidase 9 (MMP9) was induced only in the younger group. Senescence-associated ß-galactosidase (SA-ß-gal) was not detectable before or after wounding. This pilot study suggests the possibility that human cutaneous wound healing is characterized by differential expression of p21 and p53 between younger and older subjects.

Artesunate inhibits myofibroblast formation via induction of apoptosis and antagonism of pro-fibrotic gene expression in human dermal fibroblasts.

The anti-malaria drug artesunate and other chemical analogs of artemisinin have demonstrated cytostatic and cytotoxic effects in bacterial and cancer cells. Artemisinin-derived compounds have also been demonstrated to attenuate fibrosis in preclinical animal models, but the mechanisms by which this inhibition occurs are not well-understood. We investigated the effects of artesunate on the emergence of the myofibroblast, which is causally implicated in pro-fibrotic pathologies. CRL-2097 human dermal fibroblasts were analyzed for protein and transcript expression after treatment with artesunate to analyze fibroblast activation. Proliferation and apoptosis were also evaluated following treatment with artesunate in this cell line. Treatment of human dermal fibroblasts with artesunate antagonized fibroblast activation and pro-fibrotic extracellular matrix (ECM) deposition, both at basal culture conditions and when cultured in the presence of exogenous transforming growth factor-ß1 (TGF-ß1), a major pro-fibrotic cytokine. Artesunate-treated fibroblasts also demonstrated decreased proliferation and increased apoptosis. Transcript analysis by quantitative real-time polymerase chain reaction demonstrated that artesunate downregulated expression of pro-fibrotic genes including canonical myofibroblast markers, ECM genes, and several TGF-ß receptors and ligands, and upregulated expression of cell cycle inhibitors and matrix-metalloproteinases. Together, these data demonstrate that artesunate antagonizes fibroblast activation and decreases expression of pro-fibrotic genes, while also promoting myofibroblast apoptosis, suggesting that these mechanisms may be responsible in part for the anti-fibrotic effects of artesunate described previously.

Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-ß signaling results in variable activation of human dermal fibroblasts.

Fibroblast activation is a key step in the establishment of skin fibrosis induced by acute injury, and it is characterized by the differentiation of plastic resident tissue fibroblasts into contractile, extracellular matrix­secreting myofibroblasts. As fibroblast activation must be regulated in vivo, fibroblasts receive signals from the surrounding environment that initiate their fibrotic program. Thus, the present study investigated the effects of mitogen­activated protein kinase (MAPK) signaling pathways on fibroblast activation. It was demonstrated in primary human dermal fibroblasts that small molecule­mediated inhibition of extracellular signal­regulated kinase (ERK) and c­Jun N­terminal kinase (JNK) potentiated fibroblast activation, and that small molecule­mediated inhibition of p38 antagonized fibroblast activation. ERK and JNK inhibition cooperatively enhanced fibroblast activation mediated by treatment with exogenous transforming growth factor (TGF)­ß1, and p38 inhibition antagonized ERK inhibitor­mediated or JNK inhibitor­mediated fibroblast activation. Transcript analysis demonstrated that ERK and JNK inhibitor­mediated fibroblast activation was accompanied by distinct changes in the expression of TGF­ß­associated ligands and receptors, and that p38 inhibitor­mediated antagonism of fibroblast activation was accompanied by a distinct expression paradigm of TGF­ß­associated genes, including upregulation of betaglycan. ERK inhibitor­mediated and JNK inhibitor­mediated fibroblast activation was partially antagonized by small molecule­mediated inhibition of TGF­ß receptor (R)1, indicating that these mechanisms of fibroblast activation are partially dependent on TGF­ß/TGF­ßR signaling. These data collectively demonstrate and provide partial explanations of the varied effects and pathway dependencies of MAPK inhibitor­mediated effects on fibroblast activation.

Tryptophan metabolites kynurenine and serotonin regulate fibroblast activation and fibrosis.

Fibrosis is a pathological form of aberrant tissue repair, the complications of which account for nearly half of all deaths in the industrialized world. All tissues are susceptible to fibrosis under particular pathological sets of conditions. Though each type of fibrosis has characteristics and hallmarks specific to that particular condition, there appear to be common factors underlying fibrotic diseases. One of these ubiquitous factors is the paradigm of the activated myofibroblast in the promotion of fibrotic phenotypes. Recent research has implicated metabolic byproducts of the amino acid tryptophan, namely serotonin and kynurenines, in the pathology or potential pharmacologic therapy of fibrosis, in part through their effects on development of myofibroblast phenotypes. Here, we review literature underlying what is known mechanistically about the effects of these compounds and their respective pathways on fibrosis. Pharmacologic administration of kynurenine improves scarring outcomes in vivo likely not only through its well-characterized immunosuppressive properties but also via its demonstrated antagonism of fibroblast activation and of collagen deposition. In contrast, serotonin directly promotes activation of fibroblasts via activation of canonical TGF-ß signaling, and overstimulation with serotonin leads to fibrotic outcomes in vivo. Recently discovered feedback inhibition between serotonin and kynurenine pathways also reveals more information about the cellular physiology of tryptophan metabolism and may also underlie possible paradigms for anti-fibrotic therapy. Together, understanding of the effects of tryptophan metabolism on modulation of fibrosis may lead to the development of new therapeutic avenues for treatment through exploitation of these effects.

Fibroblast Growth Factor 2 as an Antifibrotic: Antagonism of Myofibroblast Differentiation and Suppression of Pro-Fibrotic Gene Expression.

Fibrosis is a pathological condition that is characterized by the replacement of dead or damaged tissue with a nonfunctional, mechanically aberrant scar, and fibrotic pathologies account for nearly half of all deaths worldwide. The causes of fibrosis differ somewhat from tissue to tissue and pathology to pathology, but in general some of the cellular and molecular mechanisms remain constant regardless of the specific pathology in question. One of the common mechanisms underlying fibroses is the paradigm of the activated fibroblast, termed the "myofibroblast," a differentiated mesenchymal cell with demonstrated contractile activity and a high rate of collagen deposition. Fibroblast growth factor 2 (FGF2), one of the members of the mammalian fibroblast growth factor family, is a cytokine with demonstrated antifibrotic activity in non-human animal, human, and in vitro models. FGF2 is highly pleiotropic and its receptors are present on many different cell types throughout the body, lending a great deal of variety to the potential mechanisms of FGF2 effects on fibrosis. However, recent reports demonstrate that a substantial contribution to the antifibrotic effects of FGF2 comes from the inhibitory effects of FGF2 on connective tissue fibroblasts, activated myofibroblasts, and myofibroblast progenitors. FGF2 demonstrates effects antagonistic towards fibroblast activation and towards mesenchymal transition of potential myofibroblast-forming cells, as well as promotes a gene expression paradigm more reminiscent of regenerative healing, such as that which occurs in the fetal wound healing response, than fibrotic resolution. With a better understanding of the mechanisms by which FGF2 alters the wound healing cascade and results in a shift away from scar formation and towards functional tissue regeneration, we may be able to further address the critical need of therapy for varied fibrotic pathologies across myriad tissue types.

FGF2-mediated attenuation of myofibroblast activation is modulated by distinct MAPK signaling pathways in human dermal fibroblasts.

BACKGROUND: Previous human and animal studies have demonstrated the ability of exogenously administered basic fibroblast growth factor (FGF2) to act as an antifibrotic agent in the skin. Though the activity of FGF2 as an anti-scarring agent is well-established for fibrotic skin wounds, the mechanisms by which FGF2 exerts these actions are not entirely understood. Canonical FGF2 signaling proceeds in part via FGFR/MAPK pathways in human dermal fibroblasts, and FGF2 has been described to prevent or reverse the fibroblast-to-myofibroblast transition, which is driven by TGFß signaling and understood to be an important step in the formation of a fibrotic scar in vivo. Thus, we set out to investigate the antagonistic effects of FGF2 on TGFß signaling as well as the broader effects of MAPK inhibition on the TGFß-mediated induction of myofibroblast gene expression. OBJECTIVE: To better understand the effects of FGF2 signaling pathways on myofibroblastic gene expression and cell phenotypes. METHODS: Human dermal fibroblasts were cultured in vitro in the presence of FGF2, TGFß, and/or MAPK inhibitors, and the effects of these agents were investigated by molecular biology techniques including qRT-PCR, immunofluorescence, Western blot, and flow cytometry. RESULTS: FGF2 inhibited TGFß-mediated fibroblast activation, resulting in more rapidly proliferating, spindle-shaped cells, compared to the more slowly proliferating, flatter TGFß-treated cells. Treatment with FGF2 also attenuated TGFß-mediated increase in expression of myofibroblast markers smooth muscle α-actin, calponin, transgelin, connective tissue growth factor, ED-A fibronectin, and collagen I. FGF2-mediated antagonism of the TGFß-mediated fibroblast-to-myofibroblast transition was reversed by small molecule inhibition of ERK or JNK, and it was potentiated by inhibition of p38. MAPK inhibition was demonstrated to have qualitatively similar effects even in the absence of exogenous FGF2, and small molecule inhibition of p38 MAPK was sufficient to attenuate TGFß-mediated fibroblast activation. CONCLUSIONS: Inhibition of select MAPK signaling pathways can reverse or potentiate anti-fibrotic FGF2 effects on human dermal fibroblasts, as well as exert their effects independently of exogenous FGF2 supplementation.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass.

A spectrum of exercise training reduces soluble Aß in a dose-dependent manner in a mouse model of Alzheimer's disease.

Physical activity has long been hypothesized to influence the risk and pathology of Alzheimer's disease. However, the amount of physical activity necessary for these benefits is unclear. We examined the effects of three months of low and high intensity exercise training on soluble Aß40 and Aß42 levels in extracellular enriched fractions from the cortex and hippocampus of young Tg2576 mice. Low (LOW) and high (HI) intensity exercise training animals ran at speeds of 15m/min on a level treadmill and 32 m/min at a 10% grade, respectively for 60 min per day, five days per week, from three to six months of age. Sedentary mice (SED) were placed on a level, non-moving, treadmill for the same duration. Soleus muscle citrate synthase activity increased by 39% in the LOW group relative to SED, and by 71% in the HI group relative to LOW, indicating an exercise training effect in these mice. Soluble Aß40 concentrations decreased significantly in an exercise training dose-dependent manner in the cortex. In the hippocampus, concentrations were decreased significantly in the HI group relative to LOW and SED. Soluble Aß42 levels also decreased significantly in an exercise training dose-dependent manner in both the cortex and hippocampus. Five proteins involved in Aß clearance (neprilysin, IDE, MMP9, LRP1 and HSP70) were elevated by exercise training with its intensity playing a role in each case. Our data demonstrate that exercise training reduces extracellular soluble Aß in the brains of Tg2576 mice in a dose-dependent manner through an up-regulation of Aß clearance.

A versatile polyacrylamide gel electrophoresis based sulfotransferase assay.

BACKGROUND: Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes. RESULTS: A universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. This assay has been successfully applied to substrates as small as alpha-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of +/- 0.2 of the actual relative mobilities. CONCLUSION: The versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.