Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

Linkage mapping of ovine microphthalmia to chromosome 23, the sheep orthologue of human chromosome 18

PURPOSE: To characterize the phenotype and map the locus responsible for autosomal recessive inherited ovine microphthalmia (OMO) in sheep. METHODS: Microphthalmia-affected lambs and their available relatives were collected in a field, and experimental matings were performed to obtain affected and normal lambs for detailed necropsy and histologic examinations. The matings resulted in 18 sheep families with 48 cases of microphthalmia. A comparative candidate gene approach was used to map the disease locus within the sheep genome. Initially, 27 loci responsible for the microphthalmia-anophthalmia phenotypes in humans or mice were selected to test for comparative linkage. Fifty flanking markers that were predicted from comparative genomic analysis to be closely linked to these genes were tested for linkage to the disease locus. After observation of statistical evidence for linkage, a confirmatory fine mapping strategy was applied by further genotyping of 43 microsatellites. RESULTS: The clinical and pathologic examinations showed slightly variable expressivity of isolated bilateral microphthalmia. The anterior eye chamber was small or absent, and a white mass admixed with cystic spaces extended from the papilla to the anterior eye chamber, while no recognizable vitreous body or lens was found within the affected eyes. Significant linkage to a single candidate region was identified at sheep chromosome 23. Fine mapping and haplotype analysis assigned the candidate region to a critical interval of 12.4 cM. This ovine chromosome segment encompasses an ancestral chromosomal breakpoint corresponding to two orthologue segments of human chromosomes 18, short and long arms. For the examined animals, we excluded the complete coding region and adjacent intronic regions of ovine TGIF1 to harbor disease-causing mutations. CONCLUSIONS: This is the first genetic localization for hereditary ovine isolated microphthalmia. It seems unlikely that a mutation in the TGIF1 gene is responsible for this disorder. The studied sheep represent a valuable large animal model for similar human ocular phenotypes.

Association between inflammatory mediators and response to inhaled nitric oxide in a model of endotoxin-induced lung injury

INTRODUCTION: Inhaled nitric oxide (INO) allows selective pulmonary vasodilation in acute respiratory distress syndrome and improves PaO2 by redistribution of pulmonary blood flow towards better ventilated parenchyma. One-third of patients are nonresponders to INO, however, and it is difficult to predict who will respond. The aim of the present study was to identify, within a panel of inflammatory mediators released during endotoxin-induced lung injury, specific mediators that are associated with a PaO2 response to INO. METHODS: After animal ethics committee approval, pigs were anesthetized and exposed to 2 hours of endotoxin infusion. Levels of cytokines, prostanoid, leucotriene and endothelin-1 (ET-1) were sampled prior to endotoxin exposure and hourly thereafter. All animals were exposed to 40 ppm INO: 28 animals were exposed at either 4 hours or 6 hours and a subgroup of nine animals was exposed both at 4 hours and 6 hours after onset of endotoxin infusion. RESULTS: Based on the response to INO, the animals were retrospectively placed into a responder group (increase in PaO2 > or = 20%) or a nonresponder group. All mediators increased with endotoxin infusion although no significant differences were seen between responders and nonresponders. There was a mean difference in ET-1, however, with lower levels in the nonresponder group than in the responder group, 0.1 pg/ml versus 3.0 pg/ml. Moreover, five animals in the group exposed twice to INO switched from responder to nonresponder and had decreased ET-1 levels (3.0 (2.5 to 7.5) pg/ml versus 0.1 (0.1 to 2.1) pg/ml, P < 0.05). The pulmonary artery pressure and ET-1 level were higher in future responders to INO. CONCLUSIONS: ET-1 may therefore be involved in mediating the response to INO.

Wound models for periodontal and bone regeneration: the role of biologic research

The ultimate goals of periodontal therapy remain the complete regeneration of those periodontal tissues lost to the destructive inflammatory-immune response, or to trauma, with tissues that possess the same structure and function, and the re-establishment of a sustainable health-promoting biofilm from one characterized by dysbiosis. This volume of Periodontology 2000 discusses the multiple facets of a transition from therapeutic empiricism during the late 1960s, toward regenerative therapies, which is founded on a clearer understanding of the biophysiology of normal structure and function. This introductory article provides an overview on the requirements of appropriate in vitro laboratory models (e.g. cell culture), of preclinical (i.e. animal) models and of human studies for periodontal wound and bone repair. Laboratory studies may provide valuable fundamental insights into basic mechanisms involved in wound repair and regeneration but also suffer from a unidimensional and simplistic approach that does not account for the complexities of the in vivo situation, in which multiple cell types and interactions all contribute to definitive outcomes. Therefore, such laboratory studies require validatory research, employing preclinical models specifically designed to demonstrate proof-of-concept efficacy, preliminary safety and adaptation to human disease scenarios. Small animal models provide the most economic and logistically feasible preliminary approaches but the outcomes do not necessarily translate to larger animal or human models. The advantages and limitations of all periodontal-regeneration models need to be carefully considered when planning investigations to ensure that the optimal design is adopted to answer the specific research question posed. Future challenges lie in the areas of stem cell research, scaffold designs, cell delivery and choice of growth factors, along with research to ensure appropriate gingival coverage in order to prevent gingival recession during the healing phase.

Processing of temporal unpredictability in human and animal amygdala

The amygdala has been studied extensively for its critical role in associative fear conditioning in animals and humans. Noxious stimuli, such as those used for fear conditioning, are most effective in eliciting behavioral responses and amygdala activation when experienced in an unpredictable manner. Here, we show, using a translational approach in mice and humans, that unpredictability per se without interaction with motivational information is sufficient to induce sustained neural activity in the amygdala and to elicit anxiety-like behavior. Exposing mice to mere temporal unpredictability within a time series of neutral sound pulses in an otherwise neutral sensory environment increased expression of the immediate-early gene c-fos and prevented rapid habituation of single neuron activity in the basolateral amygdala. At the behavioral level, unpredictable, but not predictable, auditory stimulation induced avoidance and anxiety-like behavior. In humans, functional magnetic resonance imaging revealed that temporal unpredictably causes sustained neural activity in amygdala and anxiety-like behavior as quantified by enhanced attention toward emotional faces. Our findings show that unpredictability per se is an important feature of the sensory environment influencing habituation of neuronal activity in amygdala and emotional behavior and indicate that regulation of amygdala habituation represents an evolutionary-conserved mechanism for adapting behavior in anticipation of temporally unpredictable events.

Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis

The humanized anti-alpha(4) integrin Ab Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis. Natalizumab is thought to exert its therapeutic efficacy by blocking the alpha(4) integrin-mediated binding of circulating immune cells to the blood-brain barrier (BBB). As alpha(4) integrins control other immunological processes, natalizumab may, however, execute its beneficial effects elsewhere. By means of intravital microscopy we demonstrate that natalizumab specifically inhibits the firm adhesion but not the rolling or capture of human T cells on the inflamed BBB in mice with acute experimental autoimmune encephalomyelitis (EAE). The efficiency of natalizumab to block T cell adhesion to the inflamed BBB was found to be more effective in EAE than in acute systemic TNF-alpha-induced inflammation. Our data demonstrate that alpha(4) integrin-mediated adhesion of human T cells to the inflamed BBB during EAE is efficiently blocked by natalizumab and thus provide the first direct in vivo proof of concept of this therapy in multiple sclerosis.

Epidemiology of Brucellosis and Q Fever in Linked Human and Animal Populations in Northern Togo

BACKGROUND: Although brucellosis (Brucella spp.) and Q Fever (Coxiella burnetii) are zoonoses of global importance, very little high quality data are available from West Africa. METHODS/PRINCIPAL FINDINGS: A serosurvey was conducted in Togo's main livestock-raising zone in 2011 in 25 randomly selected villages, including 683 people, 596 cattle, 465 sheep and 221 goats. Additionally, 464 transhumant cattle from Burkina Faso were sampled in 2012. The serological analyses performed were the Rose Bengal Test and ELISA for brucellosis and ELISA and the immunofluorescence assay (IFA) for Q Fever Brucellosis did not appear to pose a major human health problem in the study zone, with only 7 seropositive participants. B. abortus was isolated from 3 bovine hygroma samples, and is likely to be the predominant circulating strain. This may explain the observed seropositivity amongst village cattle (9.2%, 95%CI:4.3-18.6%) and transhumant cattle (7.3%, 95%CI:3.5-14.7%), with an absence of seropositive small ruminants. Exposure of livestock and people to C. burnetii was common, potentially influenced by cultural factors. People of Fulani ethnicity had greater livestock contact and a significantly higher seroprevalence than other ethnic groups (Fulani: 45.5%, 95%CI:37.7-53.6%; non-Fulani: 27.1%, 95%CI:20.6-34.7%). Appropriate diagnostic test cut-off values in endemic settings requires further investigation. Both brucellosis and Q Fever appeared to impact on livestock production. Seropositive cows were more likely to have aborted a foetus during the previous year than seronegative cows, when adjusted for age. This odds was 3.8 times higher (95%CI: 1.2-12.1) for brucellosis and 6.7 times higher (95%CI: 1.3-34.8) for Q Fever. CONCLUSIONS: This is the first epidemiological study of zoonoses in Togo in linked human and animal populations, providing much needed data for West Africa. Exposure to Brucella and C. burnetii is common but further research is needed into the clinical and economic impact.

Comparison of the toxicity of diesel exhaust produced by bio- and fossil diesel combustion in human lung cells in vitro

Alternative fuels are increasingly combusted in diesel- and gasoline engines and the contribution of such exhausts to the overall air pollution is on the rise. Recent findings on the possible adverse effects of biodiesel exhaust are contradictive, at least partly resulting from the various fuel qualities, engine types and different operation conditions that were tested. However, most of the studies are biased by undesired interactions between the exhaust samples and biological culture media. We here report how complete, freshly produced exhausts from fossil diesel (B0), from a blend of 20% rapeseed-methyl ester (RME) and 80% fossil diesel (B20) and from pure rapeseed methyl ester (B100) affect a complex 3D cellular model of the human airway epithelium in vitro by exposing the cells at the air–liquid interface. The induction of pro-apoptotic and necrotic cell death, cellular morphology, oxidative stress, and pro-inflammatory responses were assessed. Compared to B0 exhaust, B20 exhaust decreased oxidative stress and pro-inflammatory responses, whereas B100 exhaust, depending on exposure duration, decreased oxidative stress but increased pro-inflammatory responses. The effects are only very weak and given the compared to fossil diesel higher ecological sustainability of biodiesel, it appears that – at least RME – can be considered a valuable alternative to pure fossil diesel.

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G>A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.

Joining the dots - understanding the complex interplay between the values we place on wildlife, biodiversity conservation, human and animal health: A review.

The value of wildlife has long been ignored or under-rated. However, growing concerns about biodiversity loss and emerging diseases of wildlife origin have enhanced debates about the importance of wildlife. Wildlife-related diseases are viewed through these debates as a potential threat to wildlife conservation and domestic animal and human health. This article provides an overview of the values we place on wildlife (positive: socio-cultural, nutritional, economic, ecological; and negative: damages, health issues) and of the significance of diseases for biodiversity conservation. It shows that the values of wildlife, the emergence of wildlife diseases and biodiversity conservation are closely linked. The article also illustrates why investigations into wildlife diseases are now recognized as an integral part of global health issues. The modern One Health concept requires multi-disciplinary research groups including veterinarians, human physicians, ecologists and other scientists collaborating towards a common goal: prevention of disease emergence and preservation of ecosystems, both of which are essential to protect human life and well-being.

In Vitro and In Vivo Validation of Human and Goat Chondrocyte Labeling by Green Fluorescent Protein Lentivirus Transduction

We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, immunostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4 +/- 0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6 +/- 3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3 +/- 3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies.