HEPATIC WOUND HEALING FOLLOWING ACUTE AND CHRONIC LIVER INJURY: A POTENTIAL ROLE FOR THE HYALURONAN NETWORK

Chronic liver disease is the 12th leading cause of death in the United States and consists of a continuum of pathologies. Following initial injury, a patient develops steatosis, or excess fat accumulation. While the liver has the remarkable ability to repair itself, if injury persists, the wound healing process becomes deregulated and excess extracellular matrix (ECM) can accumulate resulting in fibrosis. If the etiologic agent is not removed, a patient can progress to cirrhosis and hepatocellular carcinoma. Despite decades of research, there are currently no effective treatments for advanced liver disease and a liver transplantation is the only treatment option for patients. There are two main approaches of developing novel therapeutics to cure liver disease. First, understanding mechanisms of liver injury can lead to potential pharmacologic targets. Second, because the liver has the remarkable capacity to heal itself, understanding the mechanisms that allow this to happen may reveal potential places that can be targeted to enhance this recovery process. This is an appealing approach because wound healing consists of mostly identical steps regardless of the injury-provoking incident and therefore could apply to liver injury of varied etiologies, and maybe even wound repair in extra-hepatic organs. Therefore, I chose to focus my dissertation research on exploring hepatic wound healing after acute and chronic liver injury. Hyaluronan (HA) is a ubiquitous, anionic glycosaminoglycan in the ECM that can hold 1,000 times its weight in water. HA, HA binding proteins, and HA receptors (the HA network) are implicated in aspects of the wound healing process in the skin, intestine, and lung. The HA network plays roles in injury, inflammation, and fibrogenesis; all primary aspects of wound healing. In fact, high basal HA levels and sustained HA induction in fetal skin are associated with the scarless wound healing that occurs after injury. Adult dermal tissue, which heals with a scar, has lower basal HA levels and a transient HA induction following injury. HA is elevated in the plasma of patients with liver disease and correlates with disease severity. Despite HA’s connection to wound healing and its use as a biomarker for liver disease, little work has been done to investigate if HA is involved in liver wound repair. We hypothesize that the HA network contributes to hepatic wound healing. Our first focus was on a role for HA itself in hepatic wound healing. HA is synthesized by one of three enzymes (HAS1, 2, or 3) and previous studies show that Has3-/- mice have decreased dextran sodium sulfate (DSS)-induced colitis and ventilator induced lung injury. We therefore, hypothesized that Has3-/- mice would be protected from liver injury and fibrosis. Using mice deficient in HAS3, wild-type mice, and carbon tetrachloride (CCl4) we induced both acute and chronic liver injury. We found that Has3-/- mice have increased injury, but also increased wound healing, including inflammation, regeneration, and matrix remodeling compared to wild-type mice after acute CCl4 exposure. This increased wound healing was associated with an increase in hepatic HA deposition and expression of the HA receptor HA mediated motility receptor (HMMR). After chronic CCl4, Has3-/- mice had increased pro-fibrotic transcripts, but no increase in fibrosis. This disconnect was attributed to increased matrix metabolism and was again associated with increased HMMR expression compared to wild-type mice. This led us to investigate a role for HMMR in hepatic wound healing. HMMR can be both intracellular and extracellular and plays a role in cell cycle progression and cell migration, important aspects of wound healing. We hypothesized that Hmmr-/- mice would have delayed wound healing following acute and chronic liver injury due to impaired hepatocyte proliferation and impaired macrophage-mediated matrix metabolism. Using CCl4, we induced both acute and chronic liver injury in Hmmr-/- and wild-type mice. Hmmr-/- mice had decreased inflammation, regeneration, and matrix remodeling despite no difference in liver injury compared to wild-type mice after acute liver injury. This was associated with decreased hepatic HA deposition. After chronic liver injury, Hmmr-/- mice had decreased pro-fibrotic transcripts but no difference in fibrosis. Similar to Has3-/- mice, this disconnect can be attributed to differences in matrix remodeling, with Hmmr-/- mice having less matrix metabolism compared to wild-type mice. My work is the first to demonstrate that HA and HMMR are involved in hepatic wound healing. The culmination of this work led to the development of a working model connecting HA and HMMR with hepatic wound healing. Following liver injury, hepatocytes and resident liver macrophages synthesize pro-inflammatory cytokines and chemokines. These cytokines then stimulate hepatic stellate cells (HSC) to synthesize and deposit HA in the injured areas of the liver, while the chemokines recruit circulating macrophages to the liver. Then, we propose two feed forward loops, the first being where the HA can increase water retention thereby increasing HSC activation by increasing mechanical stress and further increasing HA deposition. Second, we also propose a feed forward loop between HA and inflammation, where the HA contributes to inflammation and increased inflammation further increases HA synthesis by HSC. Additionally, we propose that macrophages that are being recruited to the liver express HMMR and can use the HA:HMMR interaction to localize to the necrotic area and clear tissue debris. In the case of chronic liver injury, this HA:HMMR interaction can localize scar associated macrophages to the fibrotic septae and increase matrix degradation and facilitate fibrosis resolution. Finally, the increased inflammatory microenvironment of the healing liver, and perhaps intracellular HMMR, promotes hepatocyte proliferation. Together, HA and HMMR contributes to inflammation, regeneration, and matrix remodeling to aid in the hepatic wound healing process. Taken together, our data demonstrate that the HA network, in particular HA and HMMR, is involved in both acute and chronic liver injury and wound healing. Further work should be completed to evaluate other aspects of the HA network, including other receptors and HA binding proteins. Additionally, exploring what role the HA:HMMR interaction plays in acute and chronic liver injury should be explored further

Differential effects of hyaluronan synthase 3 deficiency after acute vs chronic liver injury in mice

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Background Hyaluronan (HA) is a ubiquitous extracellular matrix (ECM) glycosaminoglycan synthesized by three different enzymes, hyaluronan synthase (HAS)1, 2, and 3. HA synthesis mediated by HAS3 promotes inflammation and is pathogenic in animal models of human lung and intestinal disease. Liver fibrosis is a common endpoint to chronic liver injury and inflammation for which there is no cure. Although plasma HA is a commonly used biomarker for liver disease, if and how HA contributes to disease pathogenesis remains unclear. Here, we tested the hypothesis that HA synthesized by HAS3 enhances inflammation and fibrosis. To test this hypothesis, we exposed wild-type or Has3−/− mice to carbon tetrachloride (CCl4) once (acute) or ten (chronic) times. Results HAS3-deficient mice exhibited increased hepatic injury and inflammatory chemokine production 48 h after acute CCl4; this was associated with a threefold reduction in plasma HA levels and alterations in the proportions of specific molecular weight HA polymer pools. Hepatic accumulation of fibrosis-associated transcripts was also greater in livers from HAS3-deficient mice compared to controls after acute CCl4 exposure. Surprisingly, fibrosis was not different between genotypes. Hepatic matrix metalloproteinase (MMP)13 mRNA and MMP13 activity was greater in livers from Has3-null mice after chronic CCl4; this was prevented by a MMP13-specific inhibitor. Collectively, these data suggest that Has3, or more likely HA produced by HAS3, limits hepatic inflammation after acute injury and attenuates MMP13-mediated matrix metabolism after chronic injury. Conclusions These data suggest that HA should be investigated further as a novel therapeutic target for acute and chronic liver disease. Electronic supplementary material The online version of this article (doi:10.1186/s13069-016-0041-5) contains supplementary material, which is available to authorized users

The impact on sleep of a multidisciplinary cognitive behavioural pain management programme: a pilot study

Background: Reduced sleep quality is a common complaint among patients with chronic pain, with 50-80% of patients reporting sleep disturbance. Improvements in pain and quality of life measures have been achieved using a multidisciplinary cognitive behavioural therapy pain management programme (CBT-PMP) that aims to recondition attitudes to pain, and improve patients' self-management of their condition. Despite its high prevalence in patients with chronic pain, there is very limited objective evidence for the effect of this intervention on sleep quality. The primary research objective is to investigate the short-term effect of a multidisciplinary CBTPMP on subjective (measured by Pittsburg Sleep Quality Index) and objective sleep quality (measured by Actigraphy) in patients with chronic pain by comparison with a control group. The secondary objectives will investigate changes in function and mood, and then explore the relationship between objective and subjective sleep quality and physical and psychological outcome measures. Methods/Design: Patients who fulfil the inclusion criteria for attendance on the multidisciplinary CBT-PMP in the Adelaide and Meath Hospital, Tallaght, Dublin and are currently listed on the PMP waiting list will be invited to participate in this pilot study. Potential patients will be screened for sleep disturbance [determined by the Pittsburgh Sleep Quality Index (PSQI)]. Those patients with a sleep disturbance (PSQI >5) will be assigned to either the intervention group (immediate treatment), or control group (deferred treatment, i.e. the PMP they are listed for is more than six months away) based on where they appear on the waiting list. Baseline measures of sleep, function, and mood will be obtained using a combination of self-report questionnaires (the Hospital Anxiety and Depression Scale, the Short Form 36 health survey, the Pittsburgh Sleep Quality Index, the Tampa Scale for Kinesiophobia), and functional outcome measures. Sleep will be measured for seven days using actigraphy (Actiwatch 7). These measures will be repeated after the four week multidisciplinary cognitive behavioural therapy pain management programme, and at a two month follow-up. The waiting list control group will be assessed at baseline, and two months later. Analysis for the primary outcome will include between group differences of subjective and objective sleep parameters from baseline to follow-up using Independent T-tests or Mann-Whitney U tests. The secondary outcomes establishing relationships between the sleep variables and physical and psychological outcome measures will be established using multiple linear regression models. Discussion: This pilot study will evaluate the impact of a multidisciplinary CBT-PMP on both subjective and objective measures of sleep in patients with chronic pain and provide guidance for a larger clinical trial. Trial Registration: Current controlled trial ISRCTN: ISRCTN7491359

Ultrasound Core Laboratory for the Household Air Pollution Intervention Network Trial: Standardized Training and Image Management for Field Studies Using Portable Ultrasound in Fetal, Lung, and Vascular Evaluations

Ultrasound Core Laboratories (UCL) are used in multicenter trials to assess imaging biomarkers to define robust phenotypes, to reduce imaging variability and to allow blinded independent review with the purpose of optimizing endpoint measurement precision. The Household Air Pollution Intervention Network, a multicountry randomized controlled trial (Guatemala, Peru, India and Rwanda), evaluates the effects of reducing household air pollution on health outcomes. Field studies using portable ultrasound evaluate fetal, lung and vascular imaging endpoints. The objective of this report is to describe administrative methods and training of a centralized clinical research UCL. A comprehensive administrative protocol and training curriculum included standard operating procedures, didactics, practical scanning and written/practical assessments of general ultrasound principles and specific imaging protocols. After initial online training, 18 sonographers (three or four per country and five from the UCL) participated in a 2 wk on-site training program. Written and practical testing evaluated ultrasound topic knowledge and scanning skills, and surveys evaluated the overall course. The UCL developed comprehensive standard operating procedures for image acquisition with a portable ultrasound system, digital image upload to cloud-based storage, off-line analysis and quality control. Pre- and post-training tests showed significant improvements (fetal ultrasound: 71% ± 13% vs. 93% ± 7%, p < 0.0001; vascular lung ultrasound: 60% ± 8% vs. 84% ± 10%, p < 0.0001). Qualitative and quantitative feedback showed high satisfaction with training (mean, 4.9 ± 0.1; scale: 1 = worst, 5 = best). The UCL oversees all stages: training, standardization, performance monitoring, image quality control and consistency of measurements. Sonographers who failed to meet minimum allowable performance were identified for retraining. In conclusion, a UCL was established to ensure accurate and reproducible ultrasound measurements in clinical research. Standardized operating procedures and training are aimed at reducing variability and enhancing measurement precision from study sites, representing a model for use of portable digital ultrasound for multicenter field studies

Resources and Geographic Access to Care for Severe Pediatric Pneumonia in Four Resource-limited Settings

Rationale: Pneumonia is the leading cause of death in children worldwide. Identifying and appropriately managing severe pneumonia in a timely manner improves outcomes. Little is known about the readiness of healthcare facilities to manage severe pediatric pneumonia in low-resource settings. Objectives: As part of the HAPIN (Household Air Pollution Intervention Network) trial, we sought to identify healthcare facilities that were adequately resourced to manage severe pediatric pneumonia in Jalapa, Guatemala (J-GUA); Puno, Peru (P-PER); Kayonza, Rwanda (K-RWA); and Tamil Nadu, India (T-IND). We conducted a facility-based survey of available infrastructure, staff, equipment, and medical consumables. Facilities were georeferenced, and a road network analysis was performed. Measurements and Main Results: Of the 350 healthcare facilities surveyed, 13% had adequate resources to manage severe pneumonia, 37% had pulse oximeters, and 44% had supplemental oxygen. Mean (±SD) travel time to an adequately resourced facility was 41 ± 19 minutes in J-GUA, 99 ± 64 minutes in P-PER, 40 ± 19 minutes in K-RWA, and 31 ± 19 minutes in T-IND. Expanding pulse oximetry coverage to all facilities reduced travel time by 44% in J-GUA, 29% in P-PER, 29% in K-RWA, and 11% in T-IND (all P < 0.001). Conclusions: Most healthcare facilities in low-resource settings of the HAPIN study area were inadequately resourced to care for severe pediatric pneumonia. Early identification of cases and timely referral is paramount. The provision of pulse oximeters to all health facilities may be an effective approach to identify cases earlier and refer them for care and in a timely manner

Unraveling the function of Arabidopsis thaliana OS9 in the endoplasmic reticulum-associated degradation of glycoproteins

In the endoplasmic reticulum, immature polypeptides coincide with terminally misfolded proteins. Consequently, cells need a well-balanced quality control system, which decides about the fate of individual proteins and maintains protein homeostasis. Misfolded and unassembled proteins are sent for destruction via the endoplasmic reticulum-associated degradation (ERAD) machinery to prevent the accumulation of potentially toxic protein aggregates. Here, we report the identification of Arabidopsis thaliana OS9 as a component of the plant ERAD pathway. OS9 is an ER-resident glycoprotein containing a mannose-6-phosphate receptor homology domain, which is also found in yeast and mammalian lectins involved in ERAD. OS9 fused to the C-terminal domain of YOS9 can complement the ERAD defect of the corresponding yeast Δyos9 mutant. An A. thaliana OS9 loss-of-function line suppresses the severe growth phenotype of the bri1-5 and bri1-9 mutant plants, which harbour mutated forms of the brassinosteroid receptor BRI1. Co-immunoprecipitation studies demonstrated that OS9 associates with Arabidopsis SEL1L/HRD3, which is part of the plant ERAD complex and with the ERAD substrates BRI1-5 and BRI1-9, but only the binding to BRI1-5 occurs in a glycan-dependent way. OS9-deficiency results in activation of the unfolded protein response and reduces salt tolerance, highlighting the role of OS9 during ER stress. We propose that OS9 is a component of the plant ERAD machinery and may act specifically in the glycoprotein degradation pathway

Fidelity and Adherence to a Liquefied Petroleum Gas Stove and Fuel Intervention during Gestation: The Multi-Country Household Air Pollution Intervention Network (HAPIN) Randomized Controlled Trial

BACKGROUND: Clean cookstove interventions can theoretically reduce exposure to household air pollution and benefit health, but this requires near-exclusive use of these types of stoves with the simultaneous disuse of traditional stoves. Previous cookstove trials have reported low adoption of new stoves and/or extensive continued traditional stove use. METHODS: The Household Air Pollution Intervention Network (HAPIN) trial randomized 3195 pregnant women in Guatemala, India, Peru, and Rwanda to either a liquefied petroleum gas (LPG) stove and fuel intervention (n = 1590) or to a control (n = 1605). The intervention consisted of an LPG stove and two initial cylinders of LPG, free fuel refills delivered to the home, and regular behavioral messaging. We assessed intervention fidelity (delivery of the intervention as intended) and adherence (intervention use) through to the end of gestation, as relevant to the first primary health outcome of the trial: infant birth weight. Fidelity and adherence were evaluated using stove and fuel delivery records, questionnaires, visual observations, and temperature-logging stove use monitors (SUMs). RESULTS: 1585 women received the intervention at a median (interquartile range) of 8.0 (5.0-15.0) days post-randomization and had a gestational age of 17.9 (15.4-20.6) weeks. Over 96% reported cooking exclusively with LPG at two follow-up visits during pregnancy. Less than 4% reported ever running out of LPG. Complete abandonment of traditional stove cooking was observed in over 67% of the intervention households. Of the intervention households, 31.4% removed their traditional stoves upon receipt of the intervention; among those who retained traditional stoves, the majority did not use them: traditional stove use was detected via SUMs on a median (interquartile range) of 0.0% (0.0%, 1.6%) of follow-up days (median follow-up = 134 days). CONCLUSIONS: The fidelity of the HAPIN intervention, as measured by stove installation, timely ongoing fuel deliveries, and behavioral reinforcement as needed, was high. Exclusive use of the intervention during pregnancy was also high

Facing the Realities of Pragmatic Design Choices in Environmental Health Studies: Experiences from the Household Air Pollution Intervention Network Trial

Objective: Household Air Pollution Intervention Network (HAPIN) investigators tested a complex, non-pharmacological intervention in four low- and middle-income countries as a strategy to mitigate household air pollution and improve health outcomes across the lifespan. Intervention households received a liquefied petroleum gas (LPG) stove, continuous fuel delivery and regular behavioral reinforcements for 18 months, whereas controls were asked to continue with usual cooking practices. While HAPIN was designed as an explanatory trial to test the efficacy of the intervention on four primary outcomes, it introduced several pragmatic aspects in its design and conduct that resemble real-life conditions. We surveyed HAPIN investigators and asked them to rank what aspects of the design and conduct they considered were more pragmatic than explanatory. Methods: We used the revised Pragmatic Explanatory Continuum Indicator Summary (PRECIS-2) to survey investigators on the degree of pragmatism in nine domains of trial design and conduct using a five-point Likert rank scale from very explanatory (1) to very pragmatic (5). We invited 103 investigators. Participants were given educational material on PRECIS-2, including presentations, papers and examples that described the use and implementation of PRECIS-2. Results: Thirty-five investigators (mean age 42 years, 51% female) participated in the survey. Overall, only 17% ranked all domains as very explanatory, with an average (±SD) rank of 3.2 ± 1.4 across domains. Fewer than 20% of investigators ranked eligibility, recruitment or setting as very explanatory. In contrast, ≥50% of investigators ranked the trial organization, delivery and adherence of the intervention and follow-up as very/rather explanatory whereas ≤17% ranked them as rather/very pragmatic. Finally, &lt;25% of investigators ranked the relevance of outcomes to participants and analysis as very/rather explanatory whereas ≥50% ranked then as rather/very pragmatic. In-country partners were more likely to rank domains as pragmatic when compared to investigators working in central coordination (average rank 3.2 vs. 2.8, respectively; Wilcoxon rank-sum p &lt; 0.001). Conclusion: HAPIN investigators did not consider their efficacy trial to be rather/very explanatory and reported that some aspects of the design and conduct were executed under real-world conditions; however, they also did not consider the trial to be overly pragmatic. Our analysis underscores the importance of using standardized tools such as PRECIS-2 to guide early discussions among investigators in the design of environmental health trials attempting to measure efficacy.</jats:p

The acceptability to patients of PhysioDirect telephone assessment and advice services; a qualitative interview study

Background: In response to long waiting lists and problems with access to primary care physiotherapy, several Primary Care Trusts (PCTs) (now Clinical Commissioning Groups CCGs) developed physiotherapy-led telephone assessment and treatment services. The Medical Research Council (MRC) funded PhysioDirect trial was a randomised control trial (RCT) in four PCTs, with a total of 2252 patients that compared this approach with usual physiotherapy care. This nested qualitative study aimed to explore the acceptability of the PhysioDirect telephone assessment and advice service to patients with musculoskeletal conditions. Methods: We conducted 57 semi-structured interviews with adults from 4 PCTs who were referred from general practice to physiotherapy with musculoskeletal conditions and were participating in the PhysioDirect trial. The Framework method was used to analyse the qualitative data. Results: The PhysioDirect service was largely viewed as acceptable although some saw it as a first step to subsequent face-to-face physiotherapy. Most participants found accessing the PhysioDirect service straightforward and smooth, and they valued the faster access to physiotherapy advice offered by the telephone service. Participants generally viewed both the PhysioDirect service and the physiotherapists providing the service as helpful. Participants’ preferences and priorities for treatment defined the acceptable features of PhysioDirect but the acceptable features were traded off against less acceptable features. Some participants felt that the PhysioDirect service was impersonal and impaired the development of a good relationship with their physiotherapist, which made the service feel remote and less valuable. Conclusion: The PhysioDirect service was broadly acceptable to participants since it provided faster access to physiotherapy advice for their musculoskeletal conditions. Participants felt that it is best placed as one method of accessing physiotherapy services, in addition to, rather than as a replacement for, more traditional face-to-face physiotherapy assessment and treatment

LPG stove and fuel intervention among pregnant women reduce fine particle air pollution exposures in three countries: Pilot results from the HAPIN trial

The Household Air Pollution Intervention Network trial is a multi-country study on the effects of a liquefied petroleum gas (LPG) stove and fuel distribution intervention on women's and children's health. There is limited data on exposure reductions achieved by switching from solid to clean cooking fuels in rural settings across multiple countries. As formative research in 2017, we recruited pregnant women and characterized the impact of the intervention on personal exposures and kitchen levels of fine particulate matter (PM2.5) in Guatemala, India, and Rwanda. Forty pregnant women were enrolled in each site. We measured cooking area concentrations of and personal exposures to PM2.5 for 24 or 48 h using gravimetric-based PM2.5 samplers at baseline and two follow-ups over two months after delivery of an LPG cookstove and free fuel supply. Mixed models were used to estimate PM2.5 reductions. Median kitchen PM2.5 concentrations were 296 μg/m3 at baseline (interquartile range, IQR: 158-507), 24 μg/m3 at first follow-up (IQR: 18-37), and 23 μg/m3 at second follow-up (IQR: 14-37). Median personal exposures to PM2.5 were 134 μg/m3 at baseline (IQR: 71-224), 35 μg/m3 at first follow-up (IQR: 23-51), and 32 μg/m3 at second follow-up (IQR: 23-47). Overall, the LPG intervention was associated with a 92% (95% confidence interval (CI): 90-94%) reduction in kitchen PM2.5 concentrations and a 74% (95% CI: 70-79%) reduction in personal PM2.5 exposures. Results were similar for each site. CONCLUSIONS: The intervention was associated with substantial reductions in kitchen and personal PM2.5 overall and in all sites. Results suggest LPG interventions in these rural settings may lower exposures to the WHO annual interim target-1 of 35 μg/m3. The range of exposure contrasts falls on steep sections of estimated exposure-response curves for birthweight, blood pressure, and acute lower respiratory infections, implying potentially important health benefits when transitioning from solid fuels to LPG