Animal Models Of Human Disease Research Articles

Sensors for Proteolytic Activity Visualization and Their Application in Animal Models of Human Diseases.

Various sensors designed for optical and photo(opto)acoustic imaging in living systems are becoming essential components of basic and applied biomedical research. Some of them including those developed for determining enzyme activity in vivo are becoming commercially available. These sensors can be used for various fluorescent signal detection methods: from whole body tomography to endoscopy with miniature cameras. Sensor molecules including enzyme-cleavable macromolecules carrying multiple quenched near-infrared fluorophores are able to deliver their payload in vivo and have long circulation time in bloodstream enabling detection of enzyme activity for extended periods of time at low doses of these sensors. In the future, more effective "activated" probes are expected to become available with optimized sensitivity to enzymatic activity, spectral characteristics suitable for intraoperative imaging of surgical field, biocompatibility and lack of immunogenicity and toxicity. New in vivo optical imaging methods such as the fluorescence lifetime and photo(opto)acoustic imaging will contribute to early diagnosis of human diseases. The use of sensors for in vivo optical imaging will include more extensive preclinical applications of experimental therapies. At the same time, the ongoing development and improvement of optical signal detectors as well as the availability of biologically inert and highly specific fluorescent probes will further contribute to the introduction of fluorescence imaging into the clinic.

An update on the tools for creating transgenic animal models of human diseases - focus on atherosclerosis.

Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.

Alpha-synuclein is highly prone to distribution in the hippocampus and midbrain in tree shrews, and its fibrils seed Lewy body-like pathology in primary neurons.

The Chinese tree shrew (TS) has many unique advantages that make it suitable for use as an experimental animal model for human disease including moderate body size, low cost of feeding, short reproductive cycle and lifespan, and close phylogenetic relationship to primates. Our previous studies have shown that TS treated with the mitochondrial inhibitor MPTP displayed classic Parkinsonian symptoms. Additionally, the structure of TS alpha-synuclein (α-syn) is highly homologous to that found in humans. Previous studies have concluded that misfolded, fibrillar α-syn is a hallmark of α-synucleinopathies. In this study, we examined the distribution and expression levels of α-syn in different TS brain regions. We also obtained recombinant TS α-syn protein to study its aggregation and cytotoxic properties in primary neurons. Our results showed that α-syn was expressed in numerous different brain regions in TS but was most abundant in the hippocampus and midbrain. The recombinant α-syn of TS displayed straight fibrils when incubated for 72 h in vitro, which is very similar to human α-syn. When exposed to primary neurons, the TS and human α-syn fibrils led to cytotoxicity and Lewy-like pathology. Our findings indicated that TS could be a potential animal model to study the pathology of α-synucleinopathies.

Treatment with PBI-4050 in patients with Alstr\xf6m syndrome: study protocol for a phase 2, single-Centre, single-arm, open-label trial

BackgroundAlström syndrome (ALMS) is a very rare autosomal recessive monogenic disorder caused by a mutation in the ALMS1 gene and characterised by childhood onset obesity, dyslipidaemia, advanced non-alcoholic fatty liver disease, diabetes and extreme insulin resistance. There is evidence of multi-organ fibrosis in ALMS and severity of the disease often leads to organ failure with associated morbidities, resulting in reduced life expectancy. There are no specific treatments for this disease, and current management consists of only symptomatic therapies. PBI-4050 is a new molecular entity with demonstrated anti-inflammatory and anti-fibrotic activities in preclinical models, including animal models of human diseases characterized by progressive fibrosis in the kidney, heart, liver and lungs. Moreover, completed Phase 2 studies in type 2 diabetes mellitus with metabolic syndrome and idiopathic pulmonary fibrosis further support the anti-inflammatory and anti-fibrotic activity of PBI-4050. Together, these data suggest that PBI-4050 has the potential to treat the pathological inflammatory and fibrotic features of ALMS. The aim of this study is to evaluate the safety and anti-inflammatory & anti-fibrotic activities of PBI-4050 in subjects with ALMS.MethodsThis is a Phase 2, single-centre, single-arm, open-label trial. A total of 18 patients with ALMS will be enrolled to receive PBI-4050 at a total daily oral dose of 800 mg for an initial 24 weeks with continuation for an additional 36 or 48 weeks. Standard assessments of safety include adverse events, clinical laboratory tests, vital signs, physical examination and electrocardiograms. Efficacy assessments include adipose tissue biopsy, hyperinsulinaemic-euglycaemic glucose clamp, adipose tissue microdialysis, liver transient elastography, liver and cardiac magnetic resonance imaging, and laboratory blood tests.DiscussionThis is the first clinical study of PBI-4050 in subjects with ALMS. Given the rarity and complexity of the disease, a single-centre, single-arm, open-label design has been chosen to maximise subject exposure and increase the likelihood of achieving our study endpoints. The results will provide valuable safety and preliminary evidence of the effects of PBI-4050 in ALMS, a rare heterogeneous disease associated with progressive fibrosis and premature mortality.Trial registrationThe trial is registered on ClinicalTrials.gov (Identifier; NCT02739217, February 2016) and European Union Drug Regulating Authorities Clinical Trials (EudraCT Number 2015–001625-16, Sept 2015).

Agricultural Animals as Biomedical Models: Occupational Health and Safety Considerations.

The use of agricultural animals in biomedical research is increasing. Their overall size and metabolic rate, organ size, longer gestation period, and other physiological similarities make them good candidates for animal models of human disease. There are a number of special considerations for use of traditional farm animals for biomedical research. Differences in physical plant infrastructure, handling equipment, training of personnel, and potential zoonoses are some of the important considerations when traditional farm animals are used in biomedical research. This article provides an overview of some of the special considerations for using traditional agricultural animals in biomedical research. With the growing need for improved translational research, it is reasonable to predict significant growth in these animal models.

Dissecting human disease with single-cell omics: application in model systems and in the clinic.

ABSTRACTProbing cellular population diversity at single-cell resolution became possible only in recent years. The popularity of single-cell ‘omic’ approaches, which allow researchers to dissect sample heterogeneity and cell-to-cell variation, continues to grow. With continuous technological improvements, single-cell omics are becoming increasingly prevalent and contribute to the discovery of new and rare cell types, and to the deciphering of disease pathogenesis and outcome. Animal models of human diseases have significantly facilitated our understanding of the mechanisms driving pathologies and resulted in the development of more efficient therapies. The application of single-cell omics to animal models improves the precision of the obtained insights, and brings single-cell technology closer to the clinical field. This Review focuses on the use of single-cell omics in cellular and animal models of diseases, as well as in samples from human patients. It also highlights the potential of these approaches to further improve the diagnosis and treatment of various pathologies, and includes a discussion of the advantages and remaining challenges in implementing these technologies into clinical practice.

Production of Genetically Engineered Porcine Embryos by Handmade Cloning.

Genetic engineering is essential to realize the full potentials of pigs both as livestock and as animal models of human disease. With the development of new genetic engineering technologies, such as the clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system, the porcine genome can be engineered with high efficiency. In this chapter, we describe a protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells, the procedures to establish single-cell-derived porcine fibroblast cell colonies carrying the desired genetic modifications, and the handmade cloning (HMC) technique to generate cloned embryos ready for embryo transfer.

Improving pregnancy outcomes in humans through studies in sheep.

Experimental studies that are relevant to human pregnancy rely on the selection of appropriate animal models as an important element in experimental design. Consideration of the strengths and weaknesses of any animal model of human disease is fundamental to effective and meaningful translation of preclinical research. Studies in sheep have made significant contributions to our understanding of the normal and abnormal development of the fetus. As a model of human pregnancy, studies in sheep have enabled scientists and clinicians to answer questions about the etiology and treatment of poor maternal, placental, and fetal health and to provide an evidence base for translation of interventions to the clinic. The aim of this review is to highlight the advances in perinatal human medicine that have been achieved following translation of research using the pregnant sheep and fetus.

Feline low-grade alimentary lymphoma: an emerging entity and a potential animal model for human disease

BackgroundLow-grade alimentary lymphoma (LGAL) is characterised by the infiltration of neoplastic T-lymphocytes, typically in the small intestine. The incidence of LGAL has increased over the last ten years and it is now the most frequent digestive neoplasia in cats and comprises 60 to 75% of gastrointestinal lymphoma cases. Given that LGAL shares common clinical, paraclinical and ultrasonographic features with inflammatory bowel diseases, establishing a diagnosis is challenging. A review was designed to summarise current knowledge of the pathogenesis, diagnosis, prognosis and treatment of feline LGAL. Electronic searches of PubMed and Science Direct were carried out without date or language restrictions.ResultsA total of 176 peer-reviewed documents were identified and most of which were published in the last twenty years. 130 studies were found from the veterinary literature and 46 from the human medicine literature. Heterogeneity of study designs and outcome measures made meta-analysis inappropriate. The pathophysiology of feline LGAL still needs to be elucidated, not least the putative roles of infectious agents, environmental factors as well as genetic events. The most common therapeutic strategy is combination treatment with prednisolone and chlorambucil, and prolonged remission can often be achieved. Developments in immunohistochemical analysis and clonality testing have improved the confidence of clinicians in obtaining a correct diagnosis between LGAL and IBD. The condition shares similarities with some diseases in humans, especially human indolent T-cell lymphoproliferative disorder of the gastrointestinal tract.ConclusionsThe pathophysiology of feline LGAL still needs to be elucidated and prospective studies as well as standardisation of therapeutic strategies are needed. A combination of conventional histopathology and immunohistochemistry remains the current gold-standard test, but clinicians should be cautious about reclassifying cats previously diagnosed with IBD to lymphoma on the basis of clonality testing. Importantly, feline LGAL could be considered to be a potential animal model for indolent digestive T-cell lymphoproliferative disorder, a rare condition in human medicine.

Base editors: a powerful tool for generating animal models of human diseases

Myriads of genetic mutations, including base substitutions, deletions, and insertions as well as chromosome structural variations, have been detected in many human diseases. Although current combination of genomics and bioinformatics has contributed greatly to understanding the genetics of these disorders, it remains challenging to ensure the causal functions of each mutation, and then to further investigate the underlying mechanism and to develop therapeutic strategies. Animal models generated by genome engineering are the key to address these issues. In this review, we will first revisit the limitation of conventional gene editing tools and mouse models generated in the past. We will then introduce a novel tool, base editors (BEs), which present a new promising approach to establish pathogenically relevant animal models. Finally, we will discuss the application of BEs in non-human primates and share our perspectives on future development of base-editing techniques.

Living inside the box: environmental effects on mouse models of human disease.

ABSTRACTThe impact of the laboratory environment on animal models of human disease, particularly the mouse, has recently come under intense scrutiny regarding both the reproducibility of such environments and their ability to accurately recapitulate elements of human environmental conditions. One common objection to the use of mice in highly controlled facilities is that humans live in much more diverse and stressful environments, which affects the expression and characteristics of disease phenotypes. In this Special Article, we review some of the known effects of the laboratory environment on mouse phenotypes and compare them with environmental effects on humans that modify phenotypes or, in some cases, have driven genetic adaptation. We conclude that the ‘boxes’ inhabited by mice and humans have much in common, but that, when attempting to tease out the effects of environment on phenotype, a controlled and, importantly, well-characterized environment is essential.

The cat as a naturally occurring model of renal interstitial fibrosis: Characterisation of primary feline proximal tubular epithelial cells and comparative pro-fibrotic effects of TGF-β1.

Chronic kidney disease (CKD) is common in both geriatric cats and aging humans, and is pathologically characterised by chronic tubulointerstitial inflammation and fibrosis in both species. Cats with CKD may represent a spontaneously occurring, non-rodent animal model of human disease, however little is known of feline renal cell biology. In other species, TGF-β1 signalling in the proximal tubular epithelium is thought to play a key role in the initiation and progression of renal fibrosis. In this study, we first aimed to isolate and characterise feline proximal tubular epithelial cells (FPTEC), comparing them to human primary renal epithelial cells (HREC) and the human proximal tubular cell line HK-2. Secondly, we aimed to examine and compare the effect of human recombinant TGF-β1 on cell proliferation, pro-apoptotic signalling and genes associated with epithelial-to-mesenchymal transition (EMT) in feline and human renal epithelial cells. FPTEC were successfully isolated from cadaverous feline renal tissue, and demonstrated a marker protein expression profile identical to that of HREC and HK-2. Exposure to TGF-β1 (0–10 ng/ml) induced a concentration-dependent loss of epithelial morphology and alterations in gene expression consistent with the occurrence of partial EMT in all cell types. This was associated with transcription of downstream pro-fibrotic mediators, growth arrest in FPTEC and HREC (but not HK-2), and increased apoptotic signalling at high concentrations of TGF- β1. These effects were inhibited by the ALK5 (TGF-β1RI) antagonist SB431542 (5 μM), suggesting they are mediated via the ALK5/TGF-β1RII receptor complex. Taken together, these results suggest that TGF-β1 may be involved in epithelial cell dedifferentiation, growth arrest and apoptosis in feline CKD as in human disease, and that cats may be a useful, naturally occurring model of human CKD.

The pertussis hypothesis: Bordetella pertussis colonization in the etiology of asthma and diseases of allergic sensitization

Decades of peer reviewed evidence demonstrate that: 1)Bordetellapertussisand pertussis toxin are potent adjuvants, inducing asthma and allergic sensitization in animal models of human disease, 2)Bordetella pertussisoften colonizes the human nasopharynx, and is well documented in highly pertussis-vaccinated populations and 3) in children, a history of whooping cough increases the risk of asthma and allergic sensitization disease. We build on these observations with six case studies and offer a pertussis-based explanation for the rapid rise in allergic disease in former East Germany following the fall of the Berlin Wall; the current asthma, peanut allergy, and anaphylaxis epidemics in the United States; the correlation between the risk of asthma and gross national income per capita by country; the lower risk of asthma and allergy in children raised on farms; and the reduced risk of atopy with increased family size and later sibling birth order. To organize the evidence for the pertussis hypothesis, we apply the Bradford Hill criteria to the association between Bordetella pertussisand asthma and allergicsensitization disease. We propose that, contrary to conventional wisdom that nasopharyngealBordetella pertussiscolonizing infections are harmless, subclinicalBordetella pertussiscolonization is an important cause of asthma and diseases of allergic sensitization.

Metagenomic comparison of the rectal microbiota between rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis).

Rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) are frequently used in establishing animal models for human diseases. To determine the differences in gut microbiota between these species, rectal swabs from 20 rhesus macaques and 21 cynomolgus macaques were collected, and the microbial composition was examined by deep sequencing of the 16S rRNA gene. We found that the rectal microbiota of cynomolgus macaques exhibited significantly higher alpha diversity than that of rhesus macaques, although the observed number of operational taxonomic units (OTUs) was almost the same. The dominant taxa at both the phylum and genus levels were similar between the two species, although the relative abundances of these dominant taxa were significantly different between them. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) showed significant differences in the functional components between the microbiota of the two species, in particular the lipopolysaccharide (LPS) synthesis proteins. The above data indicated significant differences in microbial composition and function between these two closely related macaque species, which should be taken into consideration in the future selection of these animals for disease models.

Abstract 552: Harnessing the Versatility of PLGA Nanoparticles for Targeted Cre-Mediated Recombination

The Cre/LoxP recombinase system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method enables spatial control of gene activity in almost any tissue, providing new avenues for studying gene function and establishing animal models of human diseases. A major technical advance was the development of ligand-dependent Cre recombinases that are activated by tamoxifen. While this advance has paved the way for temporally-controlled developmental studies previously deemed impractical, the generation of tissue-specific transgenic mouse lines can be time-consuming and costly. Herein, we design a ‘smart’, biocompatible, and biodegradable nanoparticle system that can be actively targeted to specific cell types in vivo through surface conjugation of antibodies. We provide proof-of-principle for successful delivery of macromolecules and plasmid DNA. Importantly, we demonstrate that these ‘smart’, tamoxifen-loaded nanoparticles can be precisely targeted to cells of interest in vivo to bind and activate Cre recombinase, resulting in tissue-specific Cre activation. This system provides a simple, yet powerful approach to induce recombination in a ubiquitious Cre system for a time- and cost-effective strategy to generate new transgenic mouse lines.

Assembly and Analysis of Unmapped Genome Sequence Reads Reveal Novel Sequence and Variation in Dogs

Dogs are excellent animal models for human disease. They have extensive veterinary histories, pedigrees, and a unique genetic system due to breeding practices. Despite these advantages, one factor limiting their usefulness is the canine genome reference (CGR) which was assembled using a single purebred Boxer. Although a common practice, this results in many high-quality reads remaining unmapped. To address this whole-genome sequence data from three breeds, Border Collie (n = 26), Bearded Collie (n = 7), and Entlebucher Sennenhund (n = 8), were analyzed to identify novel, non-CGR genomic contigs using the previously validated pseudo-de novo assembly pipeline. We identified 256,957 novel contigs and paired-end relationships together with BLAT scores provided 126,555 (49%) high-quality contigs with genomic coordinates containing 4.6 Mb of novel sequence absent from the CGR. These contigs close 12,503 known gaps, including 2.4 Mb containing partially missing sequences for 11.5% of Ensembl, 16.4% of RefSeq and 12.2% of canFam3.1+ CGR annotated genes and 1,748 unmapped contigs containing 2,366 novel gene variants. Examples for six disease-associated genes (SCARF2, RD3, COL9A3, FAM161A, RASGRP1 and DLX6) containing gaps or alternate splice variants missing from the CGR are also presented. These findings from non-reference breeds support the need for improvement of the current Boxer-only CGR to avoid missing important biological information. The inclusion of the missing gene sequences into the CGR will facilitate identification of putative disease mutations across diverse breeds and phenotypes.

Cachexia and Sarcopenia in Companion Animals: An Under‐Utilized Natural Animal Model of Human Disease

AbstractWhile laboratory small animal models of cachexia and sarcopenia are well‐suited and critical for studying mechanisms and early pre‐clinical phases for potential treatments, they are not similar enough to the human conditions to always be good predictors for results in human clinical trials. As a result, translational failures can occur when large‐scale human clinical trials are conducted on drugs, even when they appear promising in pre‐clinical studies in rodent models. What is needed is a way to more efficiently and successfully translate information gained from basic science and rodent research into human clinical trials that produce effective approved drugs. Naturally‐occurring cachexia and sarcopenia in companion animals is a more representative model of human disease that can serve as a stepping stone between basic science and human clinical trials. Many of the common diseases of humans also affect companion animals, particularly pet dogs and cats. Pet dogs and cats commonly develop heart failure, cancer, and kidney disease, as well as acute trauma or illness. The population of elderly companion animals also is increasing as pets' lifespans have become longer. As a result, both cachexia and sarcopenia are very common in companion animals. Studying these conditions in dogs and cats – either in colonies or in animal clinical trials ‐ can help to identify successful treatments that can benefit both humans and companion animals.

In vivo profiling of four centrally administered opioids for antinociception, constipation and respiratory depression: Between-colony differences in Sprague Dawley rats.

Outbred rodent stocks including Sprague Dawley rats, are known for their genetic diversity and so they are often used to develop animal models of human disease. Although between-colony differences in pharmaco-behavioural studies have been published previously, a direct head-to-head comparison study, whereby all research was performed in the same laboratory by the same experimenter utilising the supraspinal route of drug administration in the same strain of rat, is lacking. Herein, we report our head-to-head comparison study, involving assessment of antinociception, constipation and respiratory depression evoked by single bolus intracerebroventricular (ICV) doses of morphine, buprenorphine, DPDPE and U69,593 using male Sprague Dawley rats sourced from a different breeding colony (BC2) from that (BC1) used by us previously. Our data show that there are marked differences in the potency rank order for morphine and buprenorphine between rats sourced from BC2 and BC1. Although ICV morphine evoked a bell-shaped dose-response curve in the constipation test for rats from both colonies, this occurred at higher doses for rats from BC2. In conclusion, our head-to-head comparison shows considerable between-colony differences for the same rat strain, in the potency rank order of two clinically important strong opioid analgesics given by the ICV route.

Integrating data from the Investigational Medicinal Product Dossier/investigator's brochure. A new tool for translational integration of preclinical effects.

Integrating data from the Investigational Medicinal Product Dossier/investigator's brochure. A new tool for translational integration of preclinical effects.

Standardizing the Isolation, Culture‐Expansion, and Cryopreservation of Canine Umbilical‐Cord Derived Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to enable functional recovery in inflammatory injuries such as Crohn's disease and osteoarthritis. Currently, the MSC literature is focused on rodents for preclinical models of disease or clinical testing in human patients. We are interested in using the canine as a large animal model of human diseases. In this regard, the canine MSC literature lags behind human and rodent MSC, and most work on canine MSCs has focused upon bone marrow and adipose tissue‐derived with wide variation in isolation techniques. From surveying the literature and our pilot work, canine MSCs are more difficult to culture than human or rodent MSCs, have longer population doubling times, and earlier senesce. Here, a reproducible protocol for isolation and expansion of canine umbilical cord MSCs is provided. This method produces 10× greater yield per gram of tissue than other published methods. Gelatin‐coating of tissue culture plates greatly increases colony‐forming units‐fibroblast efficiency and decreases population doubling times. In addition, MSCs were able to be cultured longer before senescing, thereby increasing their cumulative population doublings. In summary, our understanding of the culture conditions needed for canine MSCs lags behind that of other species. Gelatin‐coating of tissue culture plates appears to be important for reproducible canine MSC culture. Currently, we are testing differentiation and characterization protocols to offer as standardized methods for the field.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.