Gastroesophageal reflux disease in baboons (Papio sp.): a new animal model

BACKGROUND: Gastroesophageal reflux disease (GERD) is increasingly prevalent in the human population. Current animal models require surgical or other manipulation to produce symptoms. An animal model that exhibits spontaneous GERD would provide the opportunity for much-needed research examining the susceptibility, diagnosis, and treatment of GERD. METHODS: Eight baboons (Papio hamadryas sp.) were diagnosed with GERD histopathologically using biopsies or postmortem tissues. RESULTS: The disease was characterized by a spectrum of symptoms comparable with that found in the human population. Some subjects had no gross signs of clinical disease, but were diagnosed by histopathological examination. Almost all subjects presented with at least one clinical sign of the disease. Regurgitation was the most common. CONCLUSIONS: The baboon may be a superior animal model for GERD research because it is a naturally occurring model and is anatomically and physiologically similar to humans.     

The Use of Naturally Occurring Cancer in Domestic Animals for Research into Human Cancer: General Considerations and a Review of Canine Skeletal Osteosarcoma

For many years, research into human cancer has concentrated on human patients and on artificially induced neoplasms in inbred murine hosts. Cancer, however, affects a great variety of mammals, particularly those that have been domesticated. Suchf naturally occurring neoplasms are common in dogs, cats, cattle, horses, etc., and offer fertile ground for studies relating to epidemiologyf, etiology, immunobiology, and therapy. Canine osteosarcoma is described in detail. The clinicopathologic features of this canine tumor closely approximate that of human osteosarcoma and thus make canine osteosarcoma an invaluable comparative model. Canine osteosarcoma and other naturally occurring tumors lie intermediate between the mouse models and human cancer. The use of these veterinary models in the future fabric of cancer research will broaden its base and will influence our conceptual approach to research and clinical options.     

Animal models of osteoarthritis

Animal models of osteoarthritis are used to study the pathogenesis of cartilage degeneration and to evaluate potential antiarthritic drugs for clinical use. Animal models of naturally occurring osteoarthritis (OA) occur in knee joints of guinea pigs, mice and other laboratory animal species. Transgenic models have been developed in mice. Commonly utilized surgical instability models include medial meniscal tear in guinea pigs and rats, medial or lateral partial meniscectomy in rabbits, medial partial or total meniscectomy or anterior cruciate transection in dogs. Additional models of cartilage degeneration can be induced by intra-articular iodoacetate injection or by administration of oral or parenteral quinolone antibiotics. None of these models have a proven track record of predicting efficacy in human disease since there are no agents that have been proven to provide anything other than symptomatic relief in human OA. However, agents that are active in these models are currently in clinical trials. Methodologies, gross and histopathologic features and comparisons to human disease will be discussed for the various models.     

A naturally occurring cancer with molecular connectivity to human diseases

As Jessani et al. 1 point out development of cell and animal models that accurately depict human tumorigenesis remains a major goal of cancer research. Clam cancer offers significant advantages over traditional models for genotoxic and non-genotoxic preclinical analysis of treatments for human cancers with a similar molecular basis. The naturally occurring clam model closely resembles an out-breeding, human clinical population and provides both in vitro and in vivo alternatives to those generated from inbred mouse strains or by intentional exposure to known tumor viruses. Fly and worm in vivo models for adult human somatic cell cancers do not exist because their adult somatic cells do not divide. Clam cancer is the best characterized, naturally occurring malignancy with a known molecular basis remarkably similar to those observed in several unrelated human cancers where both genotoxic and non-genotoxic strategies can restore the function of wild-type p53. To further emphasize this point of view, we here demonstrate a p53-induced, mitochondrial-directed mechanism for promoting apoptosis in the clam cancer model that is similar to one recently identified in mammals. Discerning the molecular basis for naturally occurring diseases in non-traditional models and correlating these with related molecular mechanisms responsible for human diseases is a virtually unexplored aspect of toxico-proteomics and genomics and related drug discovery.     

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.     

Humans express natural cytotoxic (NC) cell activity that is similar to murine NC cell activity

The expression of natural cytotoxic (NC) activity is well defined in mice, but poorly defined in humans. In this paper we report that humans express naturally occurring cytotoxic cell activity that recognizes and lyses murine targets that are sensitive to lysis mediated by murine NC cells, but not murine targets that are resistant to lysis by murine NC cells. We present data showing that these naturally occurring human cytotoxic cells and murine NC cells have similar lytic mechanisms. Both the human cytotoxic cells described here, and murine NC cells, use tumor necrosis factor (TNF) to mediate the lysis of sensitive targets. Moreover, targets that resist murine NC-mediated lysis by a protein synthesis-dependent post-recognitive mechanism use a similar mechanism to prevent lysis mediated by naturally occurring human cytotoxic cells. In addition to the similarity of naturally occurring human cytotoxic cells and murine NC cells in their specificity and lytic mechanism, naturally occurring human cytotoxic cells and murine NC cells are also similar in that their activity is both associated with a monocyte lineage and age independent. Taken together, these data indicate that humans express NC activity.     

Naturally occurring methylation inhibitor: DNA hypomethylation and hemoglobin synthesis in human K562 cells.

A naturally occurring methylation inhibitor isolated from rabbit liver and named methinin inhibits a number of methyltransferases. Methinin is a low-molecular-weight compound (1,400) that has an active amine group. This compound inhibits the DNA methyltransferase of human erythroleukemia cells (K562) in vitro. When the K562 cells were grown in medium containing methinin, fetal hemoglobin was produced. Small but detectable amounts of adult hemoglobin were also produced. Methinin was not toxic to these cells. The overall rate of genomic DNA methylation was reduced by 60% in cells grown in medium containing methinin. Southern blots of genomic DNA from methinin-treated cells and untreated cells hybridized to a 32P-labeled globin gene probe showed that one site in the globin gene region was hypomethylated. Methinin is a naturally occurring compound which inhibits DNA methylation both in vitro and in vivo.     

The promises and problems of linkage analysis by using the current canine genome map

There have been major advances in the canine genome map over recent years, with an agreed karyotype for all 38 pairs of autosomes and an integrated linkage-radiation-hybrid map now available. An individual dog breed represents a closed, isolated population, and many suffer genetic diseases. Some are homologs of human conditions, and dogs represent naturally occurring, potentially useful large mammal models. Other conditions may be unique to this species, but study of these offers new insights into metabolic pathways. Linkage analysis may identify a locus linked to a particular condition. Dog pedigrees facilitate these studies by being relatively more informative than human families, although planned breeding strategies to maximize the informativeness may be required. A study of a late-onset, acquired heart disease, dilated cardiomyopathy (DCM), in Newfoundland dogs highlights some of the problems. The disease occurs naturally in pedigrees within the UK in dogs kept as pets and breeding stock. Problems encountered include confirmation of the mode of inheritance and determination of phenotype. A genome-wide linkage analysis study with over 200 markers failed to detect linkage. The study indicates that more detailed linkage maps are required, and further synteny information between dog and human gained before study of diseases affecting naturally occurring families of dogs from inbred pedigrees may have maximal chance of success in order to utilize this model.     

Distribution analysis of nonsynonymous polymorphisms within the human kinase gene family

The human kinase gene family is composed of 518 genes that are involved in a diverse spectrum of physiological functions. They are also implicated in a number of diseases and encompass 10% of current drug targets. Contemporary, high-throughput sequencing efforts have identified a rich source of naturally occurring single nucleotide polymorphisms (SNPs) in kinases, a subset of which occur in the coding region of genes (cSNPs) and result in a change in the encoded amino acid sequence (nonsynonymous coding SNP; nscSNPs). What fraction of this naturally occurring variation underlies human disease is largely unknown (uDC), and much of it is assumed not to be disease causing (DC). We pursued a comprehensive computational analysis of the distribution of 1463 nscSNPs and 999 DC nscSNPs within the kinase gene family and have found that DCs are overrepresentated in the kinase catalytic domain and in receptor structures. In addition, the frequencies with which specific amino acid changes occur differ between the DCs and the uDCs, implying different biological characteristics for the two sets of human polymorphisms. Our results provide insights into the sequence and structural phenomena associated with naturally occurring kinase nscSNPs that contribute to human diseases.     

Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models

The dystrophin-glycoprotein complex (DGC) is a multisubunit complex that connects the cytoskeleton of a muscle fiber to its surrounding extracellular matrix. Mutations in the DGC disrupt the complex and lead to muscular dystrophy. There are a few naturally occurring animal models of DGC-associated muscular dystrophy (e.g. the dystrophin-deficient mdx mouse, dystrophic golden retriever dog, HFMD cat and the delta-sarcoglycan-deficient BIO 14.6 cardiomyopathic hamster) that share common genetic protein abnormalities similar to those of the human disease. However, the naturally occurring animal models only partially resemble human disease. In addition, no naturally occurring mouse models associated with loss of other DGC components are available. This has encouraged the generation of genetically engineered mouse models for DGC-linked muscular dystrophy. Not only have analyses of these mice led to a significant improvement in our understanding of the pathogenetic mechanisms for the development of muscular dystrophy, but they will also be immensely valuable tools for the development of novel therapeutic approaches for these incapacitating diseases.     

A new mouse model of Peyronie's disease: an increased expression of hypoxia-inducible factor-1 target genes during the development of penile changes

Peyronie's disease (PD) is characterized by an inflammatory response beneath the tunica albuginea with fibroblast proliferation forming a thickened fibrous plaque that may cause pain, penile curvature and erectile dysfunction. The progression of the PD plaque may eventually lead to calcification or ossification. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Research has been hampered by the lack of a universally accepted animal model. We describe an animal model of spontaneous PD in tight skin (Tsk) mice, a C57Bl/6J subline that reproduces with age important features of the human disease (fibrous plaque formation, penile bending and areas of chondroid metaplasia with heterotopic ossification). Histological analysis demonstrated an evident structural disorganization of the tunica albuginea with excessive accumulation of type I collagen. At 12 months of age, fibrous plaques with areas of chondroid metaplasia and heterotopic ossification characterized Tsk penises. The up-regulation of hypoxia-inducible factor-1 (HIF-1) leads to an increased downstream expression of HIF-1 target genes, such as TGFbeta and iNOS. These factors, together with some PDGF family members, can cause collagen deposition in Tsk penises. They can also influence chondrocyte differentiation and heterotopic bone formation. In conclusion, hypoxia, HIF-1 and HIF-1 target genes appear to play an important role in the pathogenesis of PD in Tsk mice. This mouse model that is the first example of naturally occurring model of PD in laboratory animals may aid in the identification of signalling pathways crucial for PD and should facilitate the designing and testing of new therapeutic interventions.     

Evoked Temporal Summation in Cats to Highlight Central Sensitization Related to Osteoarthritis-Associated Chronic Pain: A Preliminary Study

In cats, osteoarthritis causes significant chronic pain. Chronicity of pain is associated with changes in the central nervous system related to central sensitization, which have to be quantified. Our objectives were 1) to develop a quantitative sensory testing device in cats for applying repetitive mechanical stimuli that would evoke temporal summation; 2) to determine the sensitivity of this test to osteoarthritis-associated pain, and 3) to examine the possible correlation between the quantitative sensory testing and assessment using other pain evaluation methods. We hypothesized that mechanical sub-threshold repetitive stimuli would evoke temporal summation, and that cats with osteoarthritis would show a faster response. A blinded longitudinal study was performed in 4 non-osteoarthritis cats and 10 cats with naturally occurring osteoarthritis. Quantification of chronic osteoarthritis pain-related disability was performed over a two week period using peak vertical force kinetic measurement, motor activity intensity assessment and von Frey anesthesiometer-induced paw withdrawal threshold testing. The cats afflicted with osteoarthritis demonstrated characteristic findings consistent with osteoarthritis-associated chronic pain. After a 14-day acclimation period, repetitive mechanical sub-threshold stimuli were applied using a purpose-developed device. Four stimulation profiles of predetermined intensity, duration and time interval were applied randomly four times during a four-day period. The stimulation profiles were different (P<0.001): the higher the intensity of the stimulus, the sooner it produced a consistent painful response. The cats afflicted with osteoarthritis responded more rapidly than cats osteoarthritis free (P = 0.019). There was a positive correlation between the von Frey anesthesiometer-induced paw withdrawal threshold and the response to stimulation profiles #2 (2N/0.4 Hz) and #4 (2N/0.4 Hz): Rho_s = 0.64 (P = 0.01) and 0.63 (P = 0.02) respectively. This study is the first report of mechanical temporal summation in awake cats. Our results suggest that central sensitization develops in cats with naturally occurring osteoarthritis, providing an opportunity to improve translational research in osteoarthritis-associated chronic pain.     

Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.     

Immunopurification of the suppressor tRNA dependent rabbit beta-globin readthrough protein

In mammalian cells, the rabbit beta-globin readthrough protein is the only known example of a naturally occurring readthrough protein which does not involve a viral system. To provide an efficient means for its isolation, detection, and study, we elicited specific antibodies against this unique protein. The 22 amino acid peptide corresponding to the readthrough portion of this protein was synthesized, coupled to keyhole limpet hemocyanin, and injected into sheep. Specific antibodies to the peptide were produced as demonstrated by the enzyme-linked immunosorbent assay technique and by immunoblotting. The antibodies did not react with globin. The rabbit beta-globin readthrough protein was separated from globin and other reticulocyte proteins by polyacrylamide gel electrophoresis and visualized by silver staining or by labeling with [35S]methionine. Incorporation of [35S]methionine into the readthrough protein was significantly enhanced upon addition of an opal suppressor tRNA to reticulocyte lysates. Immunoblotting revealed that the readthrough protein also occurs in lysates without added suppressor tRNA. The antibodies were purified on an affi-gel column which had been coupled with the peptide antigen. The readthrough protein was then purified from reticulocytes by immunoaffinity chromatography and by high-performance liquid chromatography. The results provide conclusive evidence that the beta-globin readthrough protein is naturally occurring in rabbit reticulocytes.     

A method for detecting recent selection in the human genome from allele age estimates

Mutations that have recently increased in frequency by positive natural selection are an important component of naturally occurring variation that affects fitness. To identify such variants, we developed a method to test for recent selection by estimating the age of an allele from the extent of haplotype sharing at linked sites. Neutral coalescent simulations are then used to determine the likelihood of this age given the allele's observed frequency. We applied this method to a common disease allele, the hemochromatosis-associated HFE C282Y mutation. Our results allow us to reject neutral models incorporating plausible human demographic histories for HFE C282Y and one other young but common allele, indicating positive selection at HFE or a linked locus. This method will be useful for scanning the human genome for alleles under selection using the haplotype map now being constructed.     

Green tea: a new option for the prevention or control of osteoarthritis

IL-1β is a major cytokine driving the inflammatory processes leading to the pathophysiology of osteoarthritis and other inflammatory diseases. Blockade of IL-1β activity using substances such as the naturally occurring IL-1 receptor antagonist or anti-IL-1β monoclonal antibody are currently being used or tested as therapy. However, such treatments are ineffective in osteoarthritis. In a recent study, epigallocatechin-3-gallate, a green tea polyphenol, was found to be effective in reducing IL-1β-induced inflammatory cytokines, TNFα, IL-6, granulocyte-macrophage colony-stimulating factor and several chemokines from human chondrocytes. The use of green tea polyphenols may be beneficial as a therapeutic addition to biologics that control IL-1β activity by increasing effectiveness and/or reducing dosage.     

Naturally occurring human autoantibodies recognize a fetal brain antigen identified as microtubulus associated protein 1B

We have recently reported naturally occurring autoantibodies against a large fetal brain antigen (FBA). Now we describe the process of purification and identification of this particular FBA. The brains of newborn rabbits were solubilized and purified with preparative gel electrophoresis. The protein fractions were concentrated and desalted and the fractions were tested by a known positive serum. On membrane digestion of the FBA-band gave a twelve amino acid sequence that resulted in best identity score for mouse, rat and human microtubule-associated protein (MAP) 1B: a member of the microtubule-associated protein family. Monoclonal anti-MAP1B recognized a band in immunoblots of the brain homogenate and of the partially purified fractions with the same electrophoretic mobility as that recognized by a known anti-FBA positive serum. When adult rabbit brain was used as an antigen, the anti-MAP1B failed to recognize any bands on immunoblots. MAP lB has not been previously known as an autoantigen, even though many structural proteins of the neuronal cytoskeleton are known to be targets of naturally occurring autoantibodies. MAP 1B is a functionally important regulatory protein in the developing brain; thus autoantibodies against MAP1B may affect the normal development.     

Investigations of the pathogenicity of Salmonella enteritidis serotype Amsterdam following a naturally occurring infection in rats

A naturally occurring infection of an unusual Salmonella sp. was detected in research rats during routine health monitoring procedures. The organism, Salmonella enteritidis serotype Amsterdam appeared to have a low pathogenicity and did not cause clinical disease in adult rats. Experimental mouse and guinea pig inoculations suggest that the organism is more pathogenic in mice.