Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Bacterial probiotics as an aid in the control of Clostridium difficile disease in neonatal pigs.

Although Clostridium difficile infection (CDI) is a common disease in swine, there is a lack of prevention strategies. The objectives of this study were to evaluate: i) the effectiveness of Lactobacillus spp. and ii) non-toxigenic C. difficile (NTCD) as prevention for the development of CDI in piglets. Cesarean-derived piglets (N = 150) were randomly assigned to 6 groups: GROUP 1 - negative control (n = 10); GROUP 2 - NTCD only (n = 13); GROUP 3 - Lactobacillus spp. only (n = 14); GROUP 4 - positive control (challenged with toxigenic C. difficile strain) (n = 35); GROUP 5 - NTCD and challenged with the toxigenic C. difficile strain (n = 34); and GROUP 6 - Lactobacillus spp. and challenged with the toxigenic C. difficile strain (n = 44). Piglets which received NTCD showed lower prevalence of toxin-positive feces, mesocolonic edema, and microscopic lesions compared with positive control piglets. Administration of Lactobacillus spp. did not reveal clear benefits.

Probiotiques bactériens pour faciliter le contrôle de la maladie àClostridium difficilechez les porcelets néonataux. Même si l'infection par Clostridium difficile (ICD) est une maladie commune chez les porcs, il existe une absence de stratégies de prévention. Les objectifs de cette étude consistaient à évaluer: i) l'efficacité de Lactobacillus sp. et de ii) C. difficile non toxinogène (CDNT) comme méthode de prévention contre le développement de l'ICD chez les porcelets. Les porcelets délivrés par césarienne (N = 150) ont été assignés au hasard à 6 groupes: GROUPE 1 ­ groupe témoin négatif (n = 10); GROUPE 2 ­ CDNT seulement (n = 13); GROUPE 3 ­ Lactobacillus sp. seulement (n = 14); GROUPE 4 ­ groupe témoin positif (avec épreuve pour la souche toxinogène de C. difficile) (n = 35); GROUPE 5 ­ CDNT et avec épreuve pour la souche toxinogène de C. difficile (n = 34); et GROUPE 6 ­ Lactobacillus sp. et avec épreuve pour la souche toxinogène de C. difficile (n = 44). Les porcelets ayant reçu CDNT ont affiché une prévalence inférieure de fèces positives pour les toxines, de l'œdème du mésocôlon et de lésions microscopiques comparativement aux porcelets du groupe témoin positif. L'administration de Lactobacillus sp. n'a pas révélé de bienfaits évidents.(Traduit par Isabelle Vallières).

The effect of photodynamic therapy on cisterna chyli patency in rats.

OBJECTIVE: To radiographically and histologically evaluate the effects of photodynamic therapy on the cisterna chyli in rats. STUDY DESIGN: Experimental study. ANIMALS: Adult male Sprague-Dawley rats (n = 60). METHODS: Cecal lymph nodes were injected with the photodynamic compound verteporfin. A 690 nm, 500 mW diode laser was then directed at the area of the cisterna chyli for either 0, 1.5, or 3 minutes. Cisterna chyli patency was evaluated using lymphography, and histologic changes were evaluated on postoperative Days 1, 3, 5, 7, and 14. RESULTS: Histologically, minimal to marked injury to the cisternal and/or pericisternal tissues was present in all treated rats at all time periods. Radiographically, 8/20 cisternae were occluded in the 1.5-minute treatment group (including 1/4 on Day 1, 2/4 on Day 3, 3/4 on Day 5, 0/4 on Day 7, and 2/4 on Day 14), and 9/20 cisternae were occluded in the 3-minute treatment group (including 0/4 on Day 1, 1/4 on Day 3, 3/4 on Day 5, 3/4 on Day 7, and 2/4 on Day 14). There was minimal to no histologic evidence of tissue injury in control rats. All control cisternae were radiographically open. CONCLUSIONS: Further investigations into the timing of laser application and light dose, or alternative photodynamic agents are required to limit injury to adjacent tissues and to improve the effectiveness of cisternal photoablation.

Effect of age, dose and antibiotic therapy on the development of Clostridium difficile infection in neonatal piglets.

Piglet diarrhea is associated with increased pre-weaning mortality, poor growth rates, and variation in weight at weaning. Clostridium difficile is a known cause of enteric disease in neonatal piglets, yet risk factors associated with C. difficile infection in piglets are unknown. The objectives of this study were (1) to evaluate the consistency and severity of lesions in piglets challenged with C. difficile at different bacterial doses (DOSAGE experiment), (2) evaluate the use of antibiotics as a contributing risk factor in 1-day-old piglets (ANTIMICROBIAL experiment), and (3) to provide a clinical and histological evaluation of C. difficile infection in 10-day-old piglets (AGE experiment). One hundred and eleven conventional neonatal pigs were snatch farrowed and divided into experimental groups addressing the objectives. In the DOSAGE experiment, 40 1-day-old piglets were sham inoculated or challenged with varying amounts of C. difficile heat shocked spores and euthanized 72 h post infection. Results indicate a clear trend for disease development as bacterial numbers increase. In the ANTIMICROBIAL experiment, 39 1-day-old piglets were challenged and then treated with one of four different antibiotics after 16 h. No significant difference in disease development was found. Thirty-three 10-day-old piglets were given varying doses of C. difficile in the AGE experiment. Disease and lesions were reproduced in 10-day-old piglets. Combined results indicate that C. difficile dosage appears to be an important factor that influences the appearance and severity of lesions, 10-day-old pigs can develop disease associated with C. difficile, and antibiotic administration following inoculation may not be a major contributor for disease in neonatal piglets.

Effects of nanosized titanium dioxide on innate immune system of fathead minnow (Pimephales promelas Rafinesque, 1820).

Effects of nanosized (<100 nm) titanium dioxide (TiO(2)) particles on fish neutrophils and immune gene expression was investigated using the fathead minnow (Pimpehales promelas). Expanded use of TiO(2) in the cosmetic industry has increased the potential exposure risk to aquatic ecosystems and human health. Effects of nano-TiO(2) on neutrophil function of the fathead minnow was investigated using oxidative burst, neutrophil extracellular traps (NETs) release and degranulation of primary granules. The innate immune gene expression was determined with quantitative PCR (qPCR). Application of 0.1 µg mL(-1) of nano-TiO(2) in vitro stimulated oxidative burst and NET release. Intraperitoneal injection of 10 µg g(-1) of nano-TiO(2) caused a significant decrease in oxidative burst, NETs release and degranulation (21%; 11%; and 30%, decrease, respectively). Fish exposed to nano-TiO(2) for 48 h in vivo had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2 (4; 2.5; and 2 fold increase, respectively). Nano-TiO(2) has potential to interfere with the evolutionary conserved innate immune system responses, as evidenced with observed changes in gene expression and neutrophil function. This finding encourages the use of fish models in the studies of nanoparticle immunotoxicity. The lowest significant response concentration studied in vitro is four times greater than the estimated environmental concentration for TiO(2) (0.025 µg mL(-1)) causing concern about potential impact of nano-TiO(2) on aquatic animals and ecosystems.

Hydroxylated fullerenes inhibit neutrophil function in fathead minnow (Pimephales promelas Rafinesque, 1820).

Hydroxylated fullerenes act as potent inhibitors of cytochrome P450-dependent monooxygenases, and are reported to be very strong antioxidants quenching reactive oxygen species (ROS) production. Effects of nanosized hydroxylated fullerenes on fish neutrophil function and immune gene transcription was investigated using fathead minnow (Pimephales promelas). Neutrophil function assays were used to determine the effects of fullerene exposure in vitro and in vivo on oxidative burst, degranulation and extracellular trap (NETs) release, and the innate immune gene transcription was determined with quantitative PCR (qPCR). Application of fullerenes (0.2-200 microgmL(-1)in vitro) caused concentration dependent inhibition of oxidative burst and suppressed the release of NETs and degranulation of primary granules (up to 70, 40, and 50% reduction in activity compared to non-treated control, respectively). Transcription of interleukin 11 and myeloperoxidase genes was significantly increased and transcription of elastase 2 gene was significantly decreased in fish exposed to hydroxylated fullerenes for 48h in vivo (12 and 3 fold increase, and 5 fold decrease, respectively). Observed changes in gene transcription and neutrophil function indicate potential for hydroxylated fullerenes to interfere with the evolutionary conserved innate immune system responses and encourages the use of fish models in studies of nanoparticle immunotoxicity.

Echinacea-induced cytosolic Ca2+ elevation in HEK293.

BACKGROUND: With a traditional medical use for treatment of various ailments, herbal preparations of Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca2+. Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca2+ elevation is not influenced by alkamides. METHODS: A non-immune human cell line, HEK293, was chosen to evaluate E. purpurea root extracts and constituents as potential regulators of intracellular Ca2+ levels. Changes in cytosolic Ca2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor, were tested to determine the mechanism of this Ca2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity. RESULTS: A rapid transient increase in cytosolic Ca2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP3 receptor dependent. Echinacea-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca2+ elevation is sustained after the Echinacea-induced Ca2+ release from intracellular Ca2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity. CONCLUSIONS: Our data indicate that as yet unidentified constituents from Echinacea stimulate an IP3 receptor and phospholipase C mediation of cytosolic Ca2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.

Development of functional neurons from postnatal stem cells in vitro.

In order for stem cells to fulfill their clinical promise, we must understand their developmental transitions and it must be possible to control the differentiation of stem cells into specific cell fates. To understand the mechanism of the sequential restriction and multipotency of stem cells, we have established culture conditions that allow the differentiation of multipotential neural stem cells from postnatal stem cells. We used immunocytochemistry, fluorescence microscopy, and calcium imaging to demonstrate that progeny of adult rat neural stem cells develop into functional neurons that release excitatory neurotransmitters. We also found that the nontoxic heavy chain fragment of tetanus toxin, a toxin that targets neurons with high specificity, retained the specificity toward neural stem cell-derived neurons. These studies show that neural stem cells derived from adult tissues retain the potential to differentiate into functional neurons with morphological and functional properties of mature central nervous system neurons.

Effects of leptin on intracellular calcium concentrations in isolated porcine somatotropes.

Leptin, the product of the obese gene, is a protein that is secreted primarily from adipocytes. Leptin can influence the function of the pituitary gland through its action on the hypothalamus, but it can also directly act at the level of the pituitary gland. The ability of leptin to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by the application of human growth hormone releasing hormone. Leptin increased [Ca2+]i in porcine somatotropes in a dose-dependent manner. The application of 100 nM leptin for 3 min did not have a significant effect on [Ca2+]i, while a 3-min application of 1 microM leptin increased [Ca2+]i in about 50% of the somatotropes (p < 0.01). The application of a second leptin challenge (1 microM) evoked a response in only 18% of the observed somatotropes. The stimulatory effect of leptin was abolished in low calcium saline and blocked by nifedipine, an L-calcium channel blocker, suggesting an involvement of calcium channels. Pretreatment of the cultures with AG 490, a specific Janus kinase inhibitor, and with SB 203580, a mitogen-activated protein kinase (MAP kinase) inhibitor, abolished the increase in [Ca2+]i evoked by leptin. In the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, the magnitude of the increase in [Ca2+]i evoked by 1 microM leptin was not significantly changed. However, in the presence of L-NAME only 24% of the somatotropes responded to leptin, while in parallel control cultures 70% of the somatotropes responded to leptin. These results imply an involvement of Janus kinase/signal transducer and activator or transcription, MAP kinase and NOS-signaling pathways in the stimulatory effect of leptin on porcine somatotropes.

A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent.

We synthesized a MCM-41-type mesoporous silica nanosphere (MSN)-based gene transfection system, where second generation (G2) polyamidoamines (PAMAMs) were covalently attached to the surface of MSN. The G2-PAMAM-capped MSN material (G2-MSN) was used to complex with a plasmid DNA (pEGFP-C1) that encodes for an enhanced green fluorescence protein. The gene transfection efficacy, uptake mechanism, and biocompatibility of the G2-MSN system with various cell types, such as neural glia (astrocytes), human cervical cancer (HeLa), and Chinese hamster ovarian (CHO) cells, were investigated. The mesoporous structure of the MSN material allows membrane-impermeable molecules, such as pharmaceutical drugs and fluorescent dyes, to be encapsulated inside the MSN channels. The system renders the possibility to serve as a universal transmembrane carrier for intracellular drug delivery and imaging applications.