60 Years of the 3Rs symposium: Lessons learned and the road ahead.

When The Principles of Humane Experimental Technique was published in 1959, authors William Russell and Rex Burch had a modest goal: to make researchers think about what they were doing in the laboratory ­ and to do it more humanely. Sixty years later, their groundbreaking book was celebrated for inspiring a revolution in science and launching a new field: The 3Rs of alternatives to animal experimentation. On November 22, 2019, some pioneering and leading scientists and researchers in the field gathered at the Johns Hopkins Bloomberg School of Public Health in Bal­timore for the 60 Years of the 3Rs Symposium: Lessons Learned and the Road Ahead. The event was sponsored by the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), the Foundation for Chemistry Research and Initiatives, the Alternative Research & Development Foundation (ARDF), the American Cleaning Institute (ACI), the International Fragrance Association (IFRA), the Institute for In Vitro Sciences (IIVS), John "Jack" R. Fowle III, and the Society of Toxicology (SoT). Fourteen pres­entations shared the history behind the groundbreaking publication, international efforts to achieve its aims, stumbling blocks to progress, as well as remarkable achievements. The day was a tribute to Russell and Burch, and a testament to what is possible when people from many walks of life ­ science, government, and industry ­ work toward a common goal.

William Russell and Rex Burch published their book The Principles of Humane Experimental Technique in 1959. The book encouraged researchers to replace animal experiments where it was possible, to refine experiments with animals in order to reduce their suffering, and to reduce the number of animals that had to be used for experiments to the minimum. Sixty years later, a group of pioneering and leading scientists and researchers in the field gathered to share how the publi­cation came about and how the vision inspired international collaborations and successes on many different levels including new laws. The paper includes an overview of important milestones in the history of alternatives to animal experimentation.

The 35th annual advances in contrast ultrasound international bubble conference, Chicago 2021: synopsis and take-home messages.

The 35th Annual Advances in Contrast Ultrasound International Bubble Conference convened in Chicago, IL, USA, on September 30th to October 1st, 2021. It featured a range of novel research from animal studies to clinical applications in multiple organ systems, demonstrating the utility of contrast enhanced ultrasound (CEUS). A multidisciplinary group of experts on the use of CEUS, including physicians, basic scientists, engineers, and industry partners, convened to discuss cutting edge research and new applications for CEUS. The conference demonstrated the wide range of CEUS uses and potential uses, including cardiac risk stratification, sonothrombolysis, peripheral vascular reperfusion, liver and renal mass evaluation, lymphatic evaluation, sentinel node identification, and CEUS use in pediatrics. The International Contrast Ultrasound Society uses this information to continue advocating for the safe and appropriate use of CEUS.

Echocardiographic predictors of mortality and morbidity in COVID-19 disease using focused cardiovascular ultrasound.

BACKGROUND: Focused transthoracic echocardiography (fTTE) has emerged as a critical diagnostic tool during the COVID-19 pandemic, allowing for efficient cardiac imaging while minimizing staff exposure. The utility of fTTE in predicting clinical outcomes in COVID-19 remains under investigation. METHODS: We conducted a retrospective study of 2,266 hospitalized patients at Rush University Medical Center with COVID-19 infection between March and November 2020 who received a fTTE. fTTE data were analyzed for association with primary adverse outcomes (60-day mortality) and with secondary adverse outcomes (need for renal replacement therapy, need for invasive ventilation, shock, and venous thromboembolism). RESULTS: Of the 427 hospitalized patients who had a fTTE performed (mean 62 years, 43% female), 109 (26%) had died by 60 days. Among patients with an available fTTE measurement, right ventricular (RV) dilation was noted in 34% (106/309), 43% (166/386) had RV dysfunction, and 17% (72/421) had left ventricular (LV) dysfunction. In multivariable models accounting for fTTE data, RV dilation was significantly associated with 60-day mortality (OR 1.93 [CI 1.13-3.3], p = 0.016). LV dysfunction was not significantly associated with 60-day mortality (OR 0.95 [CI: 0.51-1.78], p = 0.87). CONCLUSIONS: Abnormalities in RV echocardiographic parameters are adverse prognosticators in COVID-19 disease. Patients with RV dilation experienced double the risk for 60-day mortality due to COVID-19. To our knowledge, this is the largest study to date that highlights the adverse prognostic implications of RV dilation as determined through fTTE in hospitalized COVID-19 patients.

Contrast-enhanced ultrasound in pediatric echocardiography.

The safety and benefits of cardiac contrast-enhanced ultrasound (CEUS) have been demonstrated in children and adolescents for a variety of clinical indications, including congenital heart disease. Cardiac CEUS is performed with US and the intravenous administration of ultrasound contrast agents (UCAs). It improves transthoracic echocardiography, which can be challenging in children and adults with acoustic window limitations (e.g., from obesity) and alterations in chest wall and cardiac geometry (e.g., from prior surgical procedures). Cardiac CEUS is also used to evaluate ischemia in the follow-up of congenital and acquired heart disease. In 2019, the United States Food and Drug Administration (FDA) approved a UCA for pediatric echocardiography. This article focuses on the clinical applications of UCAs in pediatric and adult echocardiography, outlining its diagnostic value, safety and potential for future applications.

Benzocaine-related methaemoglobinemia after transoesophageal echocardiography: a rare, life-threatening complication.

Methaemoglobinemia is an uncommon but potentially life-threatening complication of topical benzocaine use that requires prompt identification in patients who undergo transoesophageal echocardiography (TEE). In this case, a 21-year-old patient who had sustained a stroke with residual right-sided weakness a few days prior to presentation underwent TEE to evaluate for intracardiac shunt. She required intubation as part of her poststroke care with some instrumentation to the posterior oropharynx. Shortly after TEE, the patient experienced sudden onset respiratory distress and hypoxia that did not improve with supplemental oxygen. Chest X-ray did not reveal any acute cardiopulmonary process. Arterial blood gas co-oximetry panel with methaemoglobin level confirmed the diagnosis of methaemoglobinemia. The patient promptly received methylene blue, recovered quickly and did not have any additional episodes of hypoxia.

Expecting the unexpected: Echo laboratory preparedness in the time of COVID-19.

COVID-19 poses a unique set of challenges to the healthcare system due to its rapid spread, intensive resource utilization, and relatively high morbidity and mortality. Healthcare workers are at especially high risk of exposure given the viruses spread through close contact. Reported cardiac complications of COVID-19 include myocarditis, acute coronary syndrome, cardiomyopathy, pericardial effusion, arrhythmia, and shock. Thus, echocardiography is integral in the timely diagnosis and clinical management of COVID-19 patients. Rush University Medical Center has been at the forefront of the COVID-19 response in Illinois with high numbers of cases reported in Chicago and surrounding areas. The echocardiography laboratory at Rush University Medical Center (RUMC) proactively took numerous steps to balance the imaging needs of a busy, nearly 700-bed academic medical center while maintaining safety.

The 34th Annual Advances in Contrast Ultrasound International Bubble Conference, Chicago 2019: synopsis and take-home messages.

The 34th annual Advances in Contrast Ultrasound (ACU) International Bubble Conference convened in Chicago, IL, USA, on September 5-6, 2019 to feature new directions of research, preclinical and first-in-man clinical trials, and novel clinical applications highlighting the diversity and utility of contrast enhanced ultrasound (CEUS). An expert group comprising clinicians, engineers, basic scientists, government officials, attorneys, and industry partners convened to collaborate on cutting-edge ultrasound enhancement technology. Utilizing this information, the International Contrast Ultrasound Society (ICUS) continues to have cause to advocate for the safe and appropriate use of CEUS with expanding indications and applications.

Hypertension in older adults: Assessment, management, and challenges.

Hypertension in older adults is related to adverse cardiovascular outcomes, such as heart failure, stroke, myocardial infarction, and death. The global burden of hypertension is increasing due to an aging population and increasing prevalence of obesity, and is estimated to affect one third of the world's population by 2025. Adverse outcomes in older adults are compounded by mechanical hemodynamic changes, arterial stiffness, neurohormonal and autonomic dysregulation, and declining renal function. This review highlights the current evidence and summarizes recent guidelines on hypertension, pertaining to older adults. Management strategies for hypertension in older adults must consider the degree of frailty, increasingly complex medical comorbidities, and psycho-social factors, and must therefore be individualized. Non-pharmacological lifestyle interventions should be encouraged to mitigate the risk of developing hypertension, and as an adjunctive therapy to reduce the need for medications. Pharmacological therapy with diuretics, renin-angiotensin system blockers, and calcium channel blockers have all shown benefit on cardiovascular outcomes in older patients. Given the economic and public health burden of hypertension in the United States and globally, it is critical to address lifestyle modifications in younger generations to prevent hypertension with age.

Small Cell Lung Cancer Invading the Left Atrium With Subsequent Malignant Embolic Stroke: A Case Report and Review of Literature.

Cardiac tumors are uncommon, and the vast majority of them are metastases from extracardiac sources. Metastatic spread to the heart causes symptoms by mechanical obstruction of circulation, direct myocardial invasion, or distal embolization. We herein report a case of a 58-year-old male who presented to the hospital with multilobar intracranial embolic infarcts who was found to have small cell lung cancer (SCLC) with invasion of the left atrium and pulmonary artery resulting in malignant embolic stroke. Cerebral tumor thromboembolism from SCLC is extremely rare. This case demonstrates the thromboembolic risk associated with metastatic endoluminal cardiac tumors.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

Pulmonary hypertension: diagnosis, imaging techniques, and novel therapies.

Pulmonary hypertension (PH), defined as the elevation of mean pulmonary arterial pressure (mPAP) above 25 mmHg, has numerous causes, which the World Health Organization (WHO) has divided into five distinct categories based upon the underlying mechanism of action. This review will explore the pathophysiology, diagnostic testing, and treatment of PH. Identification of PH depends on a strong clinical suspicion, a detailed history, and a thorough physical exam. We review the evidence supporting experimental and clinical laboratory parameters for diagnosis and monitoring of PH. Transthoracic echocardiogram (TTE) is the initial screening test of choice. This review will detail specific echocardiographic techniques for the assessment and classification of PH. Furthermore, the importance of advanced imaging, including computed tomography (CT) and magnetic resonance imagining (MRI) is explored. New developments in pharmacology, percutaneous intervention, and surgical approaches are summarized. Finally, we will address the tools available to predict morbidity and mortality.

From in vivo to in vitro: The medical device testing paradigm shift.

Amid growing efforts to advance the replacement, reduction, and refinement of the use of animals in research, there is a growing recognition that in vitro testing of medical devices can be more effective, both in terms of cost and time, and also more reliable than in vivo testing. Although the technological landscape has evolved rapidly in support of these concepts, regulatory acceptance of alternative testing methods has not kept pace. Despite the acceptance by regulators of some in vitro tests (cytotoxicity, gene toxicity, and some hemocompatibility assays), many toxicity tests still rely on animals (irritation, sensitization, acute toxicity, reproductive/developmental toxicity), even where other industrial sectors have already abandoned them. Bringing about change will require a paradigm shift in current approaches to testing - and a concerted effort to generate better data on risks to human health from exposure to leachable chemicals from medical devices, and to boost confidence in the use of alternative methods to test devices. To help advance these ideas, stir debate about best practices, and coalesce around a roadmap forward, the JHU-Center for Alternatives to Animal Testing (CAAT) hosted a symposium believed to be the first gathering dedicated to the topic of in vitro testing of medical devices. Industry representatives, academics, and regulators in attendance presented evidence to support the unique strengths and challenges associated with the approaches currently in use as well as new methods under development, and drew next steps to push the field forward from their presentations and discussion.

Farm Animal Welfare and Human Health.

The paper examines the relationship between farm animal welfare, industrial farm animal production, and human health consequences. The data suggest that when the animal welfare of land-based farm animals is compromised, there are resulting significant negative human health consequences due to environmental degradation, the use of non-therapeutic levels of antibiotics for growth promotion, and the consequences of intensification. This paper accepts that even if meat and fish consumption is reduced, meat and fish will be part of the diet of the future. Industrial production modified from the current intensified systems will still be required to feed the world in 2050 and beyond. This paper identifies the concept of sustainable intensification and suggests that if farm animal welfare is improved, many of the human health consequences of intensified industrial production can be eliminated or reduced. In water-based farm animal production, many new systems are resulting in a product that actually protects the environment and can be done at industrial levels without the use of antibiotics.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

Green toxicology.

Historically, early identification and characterization of adverse effects of industrial chemicals was difficult because conventional toxicological test methods did not meet R&D needs for rapid, relatively inexpensive methods amenable to small amounts of test material. The pharmaceutical industry now front-loads toxicity testing, using in silico, in vitro, and less demanding animal tests at earlier stages of product development to identify and anticipate undesirable toxicological effects and optimize product development. The Green Chemistry movement embraces similar ideas for development of less toxic products, safer processes, and less waste and exposure. Further, the concept of benign design suggests ways to consider possible toxicities before the actual synthesis and to apply some structure/activity rules (SAR) and in silico methods. This requires not only scientific development but also a change in corporate culture in which synthetic chemists work with toxicologists. An emerging discipline called Green Toxicology (Anastas, 2012) provides a framework for integrating the principles of toxicology into the enterprise of designing safer chemicals, thereby minimizing potential toxicity as early in production as possible. Green Toxicology`s novel utility lies in driving innovation by moving safety considerations to the earliest stage in a chemical`s lifecycle, i.e., to molecular design. In principle, this field is no different than other subdisciplines of toxicology that endeavor to focus on a specific area - for example, clinical, environmental or forensic toxicology. We use the same principles and tools to evaluate an existing substance or to design a new one. The unique emphasis is in using 21st century toxicology tools as a preventative strategy to "design out" undesired human health and environmental effects, thereby increasing the likelihood of launching a successful, sustainable product. Starting with the formation of a steering group and a series of workshops, the Green Toxicology concept is currently spreading internationally and is being refined via an iterative process.

Consensus report on the future of animal-free systemic toxicity testing.

Since March 2013, animal use for cosmetics testing for the European market has been banned. This requires a renewed view on risk assessment in this field. However, in other fields as well, traditional animal experimentation does not always satisfy requirements in safety testing, as the need for human-relevant information is ever increasing. A general strategy for animal-free test approaches was outlined by the US National Research Council`s vision document for Toxicity Testing in the 21st Century in 2007. It is now possible to provide a more defined roadmap on how to implement this vision for the four principal areas of systemic toxicity evaluation: repeat dose organ toxicity, carcinogenicity, reproductive toxicity and allergy induction (skin sensitization), as well as for the evaluation of toxicant metabolism (toxicokinetics) (Fig. 1). CAAT-Europe assembled experts from Europe, America and Asia to design a scientific roadmap for future risk assessment approaches and the outcome was then further discussed and refined in two consensus meetings with over 200 stakeholders. The key recommendations include: focusing on improving existing methods rather than favoring de novo design; combining hazard testing with toxicokinetics predictions; developing integrated test strategies; incorporating new high content endpoints to classical assays; evolving test validation procedures; promoting collaboration and data-sharing of different industrial sectors; integrating new disciplines, such as systems biology and high throughput screening; and involving regulators early on in the test development process. A focus on data quality, combined with increased attention to the scientific background of a test method, will be important drivers. Information from each test system should be mapped along adverse outcome pathways. Finally, quantitative information on all factors and key events will be fed into systems biology models that allow a probabilistic risk assessment with flexible adaptation to exposure scenarios and individual risk factors.