Effects of a bioengineered allogeneic cellular construct on burn-related macrophage phenotype.

Bioengineered allogeneic cellularised constructs (BACC) exert pro-healing effects in burn wounds and skew macrophage phenotype towards a predominately reparative phenotype. However, whether BACC can modulate the phenotype of dysregulated macrophages, like those present in burn wounds, is not known. To better understand the macrophage modulatory characteristics of the BACC, primary human macrophages were polarised to the M2b phenotype, an immunosuppressive phenotype relevant to burn wounds, by simultaneously exposing macrophages to polystyrene plate-coated immunoglobulin G and the endotoxin lipopolysaccharide (LPS). The resulting macrophage phenotype upregulated both inflammatory and reparative genes, and increased secretion of the M2b marker CCL1 compared to five different in vitro macrophage phenotypes. M2b macrophages were cultured with the BACC in the presence or absence of LPS to mimic infection, which is a common occurrence in burn wounds. The BACC caused up-regulation of reparative gene sets and down-regulation of pro-inflammatory gene sets, even when LPS was present in the cell culture media. Co-cultures were maintained for 1, 3, or 5 days in the presence of LPS, and by day 1 both non-activated macrophages and M2b macrophages exhibited signs of endotoxin tolerance, as demonstrated by a reduced secretion of tumour necrosis factor α (TNFα) in response to fresh LPS stimulus. The BACC was not able to prevent endotoxin tolerance, but reparative genes were upregulated in macrophages chronically exposed to LPS. These results suggest that the BACC can promote a reparative phenotype in dysregulated macrophages relevant to the pathophysiology of burns.

Effects of a Bioengineered Allogeneic Cellularized Construct (BACC) on Primary Human Macrophage Phenotype.

The mechanisms behind the pro-healing effects of multicellular, bioengineered allogeneic cellularized constructs (BACC) are not known. Macrophages are key regulators of every phase of the wound healing process and the primary cells that mediate the response to biomaterials. It is hypothesized that cells within the BACC modulate macrophage behavior, which may contribute to the mechanism by which BACC promotes healing. To probe the influence of cells within the BACC compared to effects of the underlying collagen substrate, primary human macrophages are cultured in direct or indirect contact with BACC or with the same collagen substrate used in the BACC manufacturing. Macrophage phenotype is characterized over time via multiplex gene expression, protein secretion, multidimensional flow cytometry, and functional assays with fibroblasts and endothelial cells. The BACC causes macrophages to exhibit a predominately reparative phenotype over time compared to relevant collagen substrate controls, with multiple subpopulations expressing both pro-inflammatory and reparative markers. Conditioned media from macrophage-BACC co-cultures causes distinct effects on fibroblast and endothelial cell proliferation, migration, and network formation. Given the critical role of the reparative macrophage phenotype in wound healing, these results suggest that modulation of macrophage phenotype may be a critical part of the mechanisms behind BACC's pro-healing effects.

Ending Trans Fat-The First-Ever Global Elimination Program for a Noncommunicable Disease Risk Factor: JACC International.

Industrially produced trans fat (iTFA) is a harmful compound created as a substitute for animal and saturated fats. Estimated to cause up to 500,000 deaths per year, it is replaceable. In 2018, Resolve to Save Lives, the World Health Organization (WHO), Global Health Advocacy Incubator, and NCD Alliance partnered to achieve global trans fat elimination. The WHO Director-General called for the elimination of trans fat by 2023 through best practice policies outlined in the WHO REPLACE package. Since the accelerated global efforts in 2018, 43 countries have adopted best practice regulations protecting an additional 3.2 billion people and building momentum toward global elimination. Current coverage will prevent 66% of deaths estimated to be caused each year by trans fat in foods. Despite producing and selling iTFA-free products in many countries, companies continue to sell iTFA-containing products in unregulated markets. Global incentives, accountability mechanisms, and regional policies will help achieve the elimination goal.

Biomaterial-assisted macrophage cell therapy for regenerative medicine.

Although most tissue types are capable of some form of self-repair and regeneration, injuries that are larger than a critical threshold or those occurring in the setting of certain diseases can lead to impaired healing and ultimately loss of structure and function. The immune system plays an important role in tissue repair and must be considered in the design of therapies in regenerative medicine. In particular, macrophage cell therapy has emerged as a promising strategy that leverages the reparative roles of these cells. Macrophages are critical for successful tissue repair and accomplish diverse functions throughout all phases of the process by dramatically shifting in phenotypes in response to microenvironmental cues. Depending on their response to various stimuli, they may release growth factors, support angiogenesis, and facilitate extracellular matrix remodeling. However, this ability to rapidly shift phenotype is also problematic for macrophage cell therapy strategies, because adoptively transferred macrophages fail to maintain therapeutic phenotypes following their administration to sites of injury or inflammation. Biomaterials are a potential way to control macrophage phenotype in situ while also enhancing their retention at sites of injury. Cell delivery systems incorporated with appropriately designed immunomodulatory signals have potential to achieve tissue regeneration in intractable injuries where traditional therapies have failed. Here we explorecurrent challenges in macrophage cell therapy, especially retention and phenotype control, how biomaterials may overcome them, and opportunities for next generation strategies. Biomaterials will be an essential tool to advance macrophage cell therapy for widespread clinical applications.

Estimated health benefits, costs and cost-effectiveness of eliminating dietary industrial trans fatty acids in Kenya: cost-effectiveness analysis.

OBJECTIVES: To model the potential health gains and cost-effectiveness of a mandatory limit of industrial trans fatty acids (iTFA) in Kenyan foods. DESIGN: Multiple cohort proportional multistate life table model, incorporating existing data from the Global Burden of Disease study, pooled analyses of observational studies and peer-reviewed evidence of healthcare and policy implementation costs. SETTING: Kenya. PARTICIPANTS: Adults aged ≥20 years at baseline (n=50 million). INTERVENTION: A mandatory iTFA limit (≤2% of all fats) in the Kenyan food supply compared with a base case scenario of maintaining current trans fat intake. MAIN OUTCOME MEASURES: Averted ischaemic heart disease (IHD) events and deaths, health-adjusted life years; healthcare costs; policy implementation costs; net costs; and incremental cost-effectiveness ratio. RESULTS: Over the first 10 years, the intervention was estimated to prevent ~1900 (95% uncertainty interval (UI): 1714; 2148) IHD deaths and ~17 000 (95% UI: 15 475; 19 551) IHD events, and to save ~US$50 million (95% UI: 44; 56). The corresponding estimates over the lifespan of the model population were ~49 000 (95% UI: 43 775; 55 326) IHD deaths prevented, ~113 000 (95% UI: 100 104; 127 969) IHD events prevented and some ~US$300 million (256; 331) saved. Policy implementation costs were estimated as ~US$9 million over the first 10 years and ~US$20 million over the population lifetime. The intervention was estimated to be cost saving regardless of the time horizon. Findings were robust across multiple sensitivity analyses. CONCLUSIONS: Findings support policy action for a mandatory iTFA limit as a cost-saving strategy to avert IHD events and deaths in Kenya.

Gaining Wings to FLY: Using Drosophila Oogenesis as an Entry Point for Citizen Scientists in Laboratory Research.

Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.

Temporal Control over Macrophage Phenotype and the Host Response via Magnetically Actuated Scaffolds.

Cyclic strain generated at the cell-material interface is critical for the engraftment of biomaterials. Mechanosensitive immune cells, macrophages regulate the host-material interaction immediately after implantation by priming the environment and remodeling ongoing regenerative processes. This study investigated the ability of mechanically active scaffolds to modulate macrophage function in vitro and in vivo. Remotely actuated magnetic scaffolds enhance the phenotype of murine classically activated (M1) macrophages, as shown by the increased expression of the M1 cell-surface marker CD86 and increased secretion of multiple M1 cytokines. When scaffolds were implanted subcutaneously into mice and treated with magnetic stimulation for 3 days beginning at either day 0 or day 5 post-implantation, the cellular infiltrate was enriched for host macrophages. Macrophage expression of the M1 marker CD86 was increased, with downstream effects on vascularization and the foreign body response. Such effects were not observed when the magnetic treatment was applied at later time points after implantation (days 12-15). These results advance our understanding of how remotely controlled mechanical cues, namely, cyclic strain, impact macrophage function and demonstrate the feasibility of using mechanically active nanomaterials to modulate the host response in vivo.

Cost assessment of a program for laboratory testing of plasma trans-fatty acids in Thailand.

Objectives: Intake of trans fatty acids (TFA) increases the risk of cardiovascular disease. Assessment of TFA exposure in the population is key for determining TFA burden and monitoring change over time. One approach for TFA monitoring is measurement of TFA levels in plasma. Understanding costs associated with this approach can facilitate program planning, implementation and scale-up. This report provides an assessment of costs associated with a pilot program to measure plasma TFA levels in Thailand. Study design: Cost analysis in a laboratory facility in Thailand. Methods: We defined three broad cost modules: laboratory, personnel, and facility costs, which were further classified into sub-components and into fixed and variable categories. Costs were estimated based on the number of processed plasma samples (100-2700 in increments of 50) per year over a certain number of years (1-5), in both USD and Thai Baht. Total cost and average costs per sample were estimated across a range of samples processed. Results: The average cost per sample of analyzing 900 samples annually over 5 years was estimated at USD186. Laboratory, personnel, and facility costs constitute 67%, 23%, and 10% of costs, respectively. The breakdown across fixed costs, such as laboratory instruments and personnel, and variable costs, such as chemical supplies, was 60% and 40%, respectively. Average costs decline as more samples are processed: the cost per sample for analyzing 100, 500, 1500, and 2500 samples per year over 5 years is USD1351, USD301, USD195; and USD177, respectively. Conclusions: Laboratory analysis of plasma TFA levels has high potential for economies of scale, encouraging a long-term approach to TFA monitoring initiatives, particularly in countries that already maintain national biometric repositories.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells.

BACKGROUND/AIM: We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. MATERIALS AND METHODS: Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. RESULTS: High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. CONCLUSION: PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.

Earlier Outbreak Detection-A Generic Model and Novel Methodology to Guide Earlier Detection Supported by Data From Low- and Mid-Income Countries.

Infectious disease outbreaks can have significant impact on individual health, national economies, and social well-being. Through early detection of an infectious disease, the outbreak can be contained at the local level, thereby reducing adverse effects on populations. Significant time and funding have been invested to improve disease detection timeliness. However, current evaluation methods do not provide evidence-based suggestions or measurements on how to detect outbreaks earlier. Key conditions for earlier detection and their influencing factors remain unclear and unmeasured. Without clarity about conditions and influencing factors, attempts to improve disease detection remain ad hoc and unsystematic. Methods: We developed a generic five-step disease detection model and a novel methodology to use for data collection, analysis, and interpretation. Data was collected in two workshops in Southeast Europe (n = 33 participants) and Southern and East Africa (n = 19 participants), representing mid- and low-income countries. Through systematic, qualitative, and quantitative data analyses, we identified key conditions for earlier detection and prioritized factors that influence them. As participants joined a workshop format and not an experimental setting, no ethics approval was required. Findings: Our analyses suggest that governance is the most important condition for earlier detection in both regions. Facilitating factors for earlier detection are risk communication activities such as information sharing, communication, and collaboration activities. Impeding factors are lack of communication, coordination, and leadership. Interpretation: Governance and risk communication are key influencers for earlier detection in both regions. However, inadequate technical capacity, commonly assumed to be a leading factor impeding early outbreak detection, was not found a leading factor. This insight may be used to pinpoint further improvement strategies.

Priority Actions to Advance Population Sodium Reduction.

High sodium intake is estimated to cause approximately 3 million deaths per year worldwide. The estimated average sodium intake of 3.95 g/day far exceeds the recommended intake. Population sodium reduction should be a global priority, while simultaneously ensuring universal salt iodization. This article identifies high priority strategies that address major sources of sodium: added to packaged food, added to food consumed outside the home, and added in the home. To be included, strategies needed to be scalable and sustainable, have large benefit, and applicable to one of four measures of effectiveness: (1) Rigorously evaluated with demonstrated success in reducing sodium; (2) suggestive evidence from lower quality evaluations or modeling; (3) rigorous evaluations of similar interventions not specifically for sodium reduction; or (4) an innovative approach for sources of sodium that are not sufficiently addressed by an existing strategy. We identified seven priority interventions. Four target packaged food: front-of-pack labeling, packaged food reformulation targets, regulating food marketing to children, and taxes on high sodium foods. One targets food consumed outside the home: food procurement policies for public institutions. Two target sodium added at home: mass media campaigns and population uptake of low-sodium salt. In conclusion, governments have many tools to save lives by reducing population sodium intake.

Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells.

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Drivers of earlier infectious disease outbreak detection: a systematic literature review.

BACKGROUND: The early detection of infectious disease outbreaks can reduce the ultimate size of the outbreak, with lower overall morbidity and mortality due to the disease. Numerous approaches to the earlier detection of outbreaks exist, and methods have been developed to measure progress on timeliness. Understanding why these surveillance approaches work and do not work will elucidate key drivers of early detection, and could guide interventions to achieve earlier detection. Without clarity about the conditions necessary for earlier detection and the factors influencing these, attempts to improve surveillance will be ad hoc and unsystematic. METHODS: A systematic review was conducted using the PRISMA framework (Preferred Reporting Items for Systematic Reviews and Meta-analyses) to identify research published between January 1, 1990 and December 31, 2015 in the English language. The MEDLINE (PubMed) database was searched. Influencing factors were organized according to a generic five-step infectious disease detection model. RESULTS: Five studies were identified and included in the review. These studies evaluated the effect of electronic-based reporting on detection timeliness, impact of laboratory agreements on timeliness, and barriers to notification by general practitioners. Findings were categorized as conditions necessary for earlier detection and factors that influence whether or not these conditions can be in place, and were organized according to the detection model. There is some evidence on reporting, no evidence on assessment, and speculation about local level recognition. CONCLUSION: Despite significant investment in early outbreak detection, there is very little evidence with respect to factors that influence earlier detection. More research is needed to guide intervention planning.

Magnetic Nanoparticle-Mediated Targeting of Cell Therapy Reduces In-Stent Stenosis in Injured Arteries.

Although drug-eluting stents have dramatically reduced the recurrence of restenosis after vascular interventions, the nonselective antiproliferative drugs released from these devices significantly delay reendothelialization and vascular healing, increasing the risk of short- and long-term stent failure. Efficient repopulation of endothelial cells in the vessel wall following injury may limit complications, such as thrombosis, neoatherosclerosis, and restenosis, through reconstitution of a luminal barrier and cellular secretion of paracrine factors. We assessed the potential of magnetically mediated delivery of endothelial cells (ECs) to inhibit in-stent stenosis induced by mechanical injury in a rat carotid artery stent angioplasty model. ECs loaded with biodegradable superparamagnetic nanoparticles (MNPs) were administered at the distal end of the stented artery and localized to the stent using a brief exposure to a uniform magnetic field. After two months, magnetic localization of ECs demonstrated significant protection from stenosis at the distal part of the stent in the cell therapy group compared to both the proximal part of stent in the cell therapy group and the control (stented, nontreated) group: 1.7-fold (p < 0.001) less reduction in lumen diameter as measured by B-mode and color Doppler ultrasound, 2.3-fold (p < 0.001) less reduction in the ratios of peak systolic velocities as measured by pulsed wave Doppler ultrasound, and 2.1-fold (p < 0.001) attenuation of stenosis as determined through end point morphometric analysis. The study thus demonstrates that magnetically assisted delivery of ECs is a promising strategy for prevention of vessel lumen narrowing after stent angioplasty procedure.

Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction.

The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.