scholarly journals Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256143
Author(s):  
Pascal Hoffmann ◽  
Nadine Schnepel ◽  
Marion Langeheine ◽  
Katrin Künnemann ◽  
Guntram A. Grassl ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Cholera Toxin ◽  
Short Circuit ◽  
Animal Health ◽  
Animal Experiments ◽  
Chloride Secretion ◽  
Farm Animals ◽  
Laboratory Animals ◽  
Species Specific

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

scholarly journals Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0257824
Author(s):  
Pascal Hoffmann ◽  
Marion Burmester ◽  
Marion Langeheine ◽  
Ralph Brehm ◽  
Michael T. Empl ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Short Circuit ◽  
Functional Model ◽  
Chloride Secretion ◽  
Bacterial Toxins ◽  
Secretory Diarrhea ◽  
Laboratory Animals ◽  
Structural Level ◽  
Heat Labile ◽  
Physiological Properties

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

scholarly journals State of the art on lung organoids in mammals

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Fabienne Archer ◽  
Alexandra Bobet-Erny ◽  
Maryline Gomes
Keyword(s):  
Lung Development ◽  
One Health ◽  
State Of The Art ◽  
Animal Health ◽  
In Vitro Models ◽  
Cancer Genetic ◽  
3 Dimensional ◽  
The One ◽  
Species Specific

AbstractThe number and severity of diseases affecting lung development and adult respiratory function have stimulated great interest in developing new in vitro models to study lung in different species. Recent breakthroughs in 3-dimensional (3D) organoid cultures have led to new physiological in vitro models that better mimic the lung than conventional 2D cultures. Lung organoids simulate multiple aspects of the real organ, making them promising and useful models for studying organ development, function and disease (infection, cancer, genetic disease). Due to their dynamics in culture, they can serve as a sustainable source of functional cells (biobanking) and be manipulated genetically. Given the differences between species regarding developmental kinetics, the maturation of the lung at birth, the distribution of the different cell populations along the respiratory tract and species barriers for infectious diseases, there is a need for species-specific lung models capable of mimicking mammal lungs as they are of great interest for animal health and production, following the One Health approach. This paper reviews the latest developments in the growing field of lung organoids.

188 DEVELOPMENT OF AN EFFECTIVE WHOLE-OVARY PERFUSION SYSTEM

2015 ◽  
Vol 27 (1) ◽  
pp. 185
Author(s):  
S. Maffei ◽  
G. Galeati ◽  
G. Pennarossa ◽  
T. A. L. Brevini ◽  
G. Gandolfi
Keyword(s):  
Cell Proliferation ◽  
Ex Vivo ◽  
Animal Health ◽  
Basal Medium ◽  
Laboratory Animals ◽  
Large Animal ◽  
Perfusion System ◽  
Whole Ovaries

The different structures of a mammalian ovary require complex 3-dimensional interactions to function properly. It is difficult to access the ovary in vivo and to study its physiology in vitro, it is necessary to dissect its different parts and culture them individually. Although informative, this approach prevents the understanding of the role played by their interactions. Perfusion systems are available for ovaries of laboratory animals while organs of larger species have been maintained in culture only for a few hours. This has prompted us to develop a system that can preserve the function of a whole sheep ovary for a few days ex vivo so that it is available for analysis in controlled conditions. Twenty-four sheep ovaries were collected at the local abattoir; 18 were assigned randomly to 3 experimental groups (media A, B, and C) and 6 were immediately fixed in 10% formaldehyde and used as fresh controls. Whole ovaries were cultured for up to 4 days using a semi-open perfusion system. Organs were perfused through the ovarian artery, at a flow rate of 1.5 mL min–1 with basal medium (M199, 25 mM HEPES, 2 mM l-glutamine and 100 µg mL–1 antibiotic-antimycotic solution) supplemented with 0.4% fatty acid free BSA (medium A); or 0.4% BSA heat shock fraction (medium B); or 10% FBS, 50 ng mL–1 IGF-1, and 50 mg bovine insulin (medium C). Ovaries were stimulated with FSH (Folltropin®-V, Bioniche Animal Health Inc., Belleville, Ontario, Canada) changing medium in a pulsatile manner (1 mg mL–1 for 2 h; 0.5 mg mL–1 for 2 h; 0 mg mL–1 for 20 h), with the same cycle repeated each day of culture. At every change, aliquots were collected for oestradiol (E2) and progesterone (P4) quantification. After culture, ovaries were examined for follicular morphology, cell proliferation, and apoptotic rate. Statistical analysis was performed using one-way ANOVA (SPSS 20, IBM, Armonk, NY, USA). In media A and B, all morphological parameters showed a small but significant decrease compared to fresh control, only after 3 days of culture. The different BSA in medium B did not affect follicle morphology but significantly increased cell proliferation (medium A, 28.59 ± 3.26%; medium B, 32.04 ± 2.67%) and decreased apoptosis (medium A, 32.51 ± 5.92%; medium B, 24.55 ± 2.55%). In both media, steroid concentration increased after FSH pulses (E2 range 1.95–10.50 pg mL–1; P4 range 0.34–3.08 ng mL–1), reaching levels similar to those measurable in peripheral plasma. The presence of FBS, IGF-1, and insulin in medium C allowed extension of the culture period to 4 days with a percentage of intact follicles comparable to that observed after 3 days in media A and B. Moreover, proliferation rates were comparable to fresh controls. Steroid pattern changed with P4 values dropping close to zero (range 0.03–1.18 ng mL–1) and E2 level (range 23.59–94.98 pg mL–1) increasing 10-fold, achieving a concentration similar to that measured in the ovarian vein around oestrous. Our data indicate that it is possible to support viability of large animal whole ovaries for up to 4 days, providing a physiologically relevant model for studying ovarian functions in vitro. Research was supported by AIRC IG 10376 and by the Carraresi Foundation.

An Investigation of the Role of Possible Neural Mechanisms in Cholera Toxin-Induced Secretion in Rabbit Ileal Mucosa in Vitro

1989 ◽  
Vol 77 (2) ◽  
pp. 161-166 ◽  
Author(s):  
K. J. Moriarty ◽  
N. B. Higgs ◽  
M. Woodford ◽  
L. A. Turnberg
Keyword(s):  
Cholera Toxin ◽  
Fluid Transport ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Rabbit Ileum ◽  
Ileal Mucosa

1. Cholera toxin stimulates intestinal secretion in vitro by activation of mucosal adenylate cyclase. However, it has been proposed that cholera toxin promotes secretion in vivo mainly through an indirect mechanism involving enteric neural reflexes. 2. We examined this hypothesis further by studying the influence of neuronal blockade on cholera toxin-induced changes in fluid transport across rabbit ileum in vitro. Mucosa, stripped of muscle layers, was mounted in flux chambers and luminal application of crude cholera toxin (2 μg/ml) caused a delayed but sustained rise in the short-circuit current, electrical potential difference and Cl− secretion. Pretreatment with the nerve-blocking drug, tetrodotoxin (5 × 10−6 mol/l serosal side), failed to influence the secretory response to cholera toxin, and addition of tetrodotoxin at the peak response to cholera toxin also had no effect. 3. That tetrodotoxin could block neurally mediated secretagogues was confirmed by the demonstration that the electrical responses to neurotensin (10−7 mol/l and 10−8 mol/l) were blocked by tetrodotoxin (5 × 10−6 mol/l). Furthermore, the response to cholera toxin of segments of ileum, which included the myenteric, submucosal and mucosal nerve plexuses, was not inhibited by tetrodotoxin. 4. We conclude that cholera toxin-induced secretion in rabbit ileum in vitro is not mediated via a neurological mechanism.

scholarly journals Prawo o zdrowiu zwierząt

2020 ◽  
pp. 41-52
Author(s):  
Izabela Lipińska
Keyword(s):  
Health Status ◽  
Infectious Diseases ◽  
Animal Health ◽  
Legal Regulation ◽  
Farm Animals ◽  
Animal Disease ◽  
Fully Integrated ◽  
Animal Disease Control ◽  
The Subject ◽  
Health Legislation

The subject of the article is the issues related to the implementation of EU legal solutions concerning the protection of the health of farm animals, while the purpose of the considerations is to evaluate the legal regulation adopted to implement the package of measures aimed at animal health that have been in place since 2013, and propose the direction of necessary changes in the national legislation in this area. The new animal health legislation entails the amendment or repeal of a number of national provisions on animal disease control, in particular those which were transpositions of directives and EU decisions now repealed. The legal instruments provided for in the regulation are expected to contribute to the achievement of a fully integrated internal market and to prevent the spread of infectious diseases, provided that the existing animal health status is maintained as far as possible.

Isolated hemoperfused heart model of slaughterhouse pigs

2001 ◽  
Vol 24 (4) ◽  
pp. 215-221 ◽  
Author(s):  
D. Modersohn ◽  
S. Eddicks ◽  
C. Grosse-Siestrup ◽  
I. Ast ◽  
S. Holinski ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Heart Function ◽  
Animal Experiments ◽  
Laboratory Animals ◽  
Coronary Perfusion ◽  
Balloon Catheters ◽  
Wide Range ◽  
Infusion Of Norepinephrine

A model of hemoperfused slaughterhouse pighearts is described providing a wide range of applications which leads to a reduction in animal experiments. The size of a pigheart, heart rate, coronary perfusion, metabolism, etc. are more comparable to conditions in patients than those in hearts of small laboratory animals. Global heart function can be assessed either by measuring stroke volume, ejection fraction, Emaxetc. in the working model or by measuring intraventricular pressure with balloon catheters in the isovolumetric model. Regional cardiac function can be measured by sonomicrometry and ischemic and non-ischemic areas can be compared. Local metabolic changes are measurable as well with microdialysis. Cardiac function can be kept on any given functional level by infusion of norepinephrine in spite of the fact that functional parameters are lower without adrenergic drive in vitro than in vivo. Stable heart function can be maintained for several hours with only 500 to 1000 ml of blood because the blood is permanently regenerated by a special dialysis system. This model can be applied in many research projects dealing with reperfusion injuries, inotropic, antiarrhythmic or arrhythmogenic effects of certain drugs, immunological rejection, evaluation of imaging systems (NMR, echocardiography etc.) or cardiac assist devices.

A novel plant-derived inhibitor of cAMP-mediated fluid and chloride secretion

1999 ◽  
Vol 276 (1) ◽  
pp. G58-G63 ◽  
Author(s):  
S. E. Gabriel ◽  
S. E. Davenport ◽  
R. J. Steagall ◽  
V. Vimal ◽  
T. Carlson ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Ussing Chamber ◽  
Fluid Secretion ◽  
Chloride Secretion ◽  
Inhibitory Effect ◽  
Adult Mice ◽  
Dependent Inhibition ◽  
Chamber Studies

We have identified an agent (SP-303) that shows efficacy against in vivo cholera toxin-induced fluid secretion and in vitro cAMP-mediated Cl−secretion. Administration of cholera toxin to adult mice results in an increase in fluid accumulation (FA) in the small intestine (FA ratio = 0.63 vs. 1.86 in control vs. cholera toxin-treated animals, respectively). This elevation in FA induced by cholera toxin was significantly reduced (FA ratio = 0.70) in animals treated with a 100 mg/kg dose of SP-303 at the same time as the cholera treatment. Moreover, when SP-303 was administered 3 h after cholera toxin, a dose-dependent inhibition of FA levels was observed with a half-maximal inhibitory dose of 10 mg/kg. In Ussing chamber studies of Caco-2 or T84 monolayer preparations, SP-303 had a significant effect on both basal current and forskolin-stimulated Cl−current. SP-303 also induced an increase in resistance that paralleled the observed decrease in current. These data suggest that SP-303 has an inhibitory effect on cAMP-mediated Cl−and fluid secretion. Thus SP-303 may prove to be a useful broad-spectrum antidiarrheal agent.

scholarly journals Composing the Early Embryonic Microenvironment: Physiology and Regulation of Oviductal Secretions

2019 ◽  
Vol 21 (1) ◽  
pp. 223 ◽  
Author(s):  
Marie Saint-Dizier ◽  
Jennifer Schoen ◽  
Shuai Chen ◽  
Charles Banliat ◽  
Pascal Mermillod
Keyword(s):  
Embryonic Development ◽  
Farm Animals ◽  
Meat Production ◽  
Mammalian Embryos ◽  
Production Techniques ◽  
Species Specific ◽  
Molecular Components ◽  
Oviductal Fluid

The oviductal fluid is the first environment experienced by mammalian embryos at the very beginning of life. However, it has long been believed that the oviductal environment was not essential for proper embryonic development. Successful establishment of in vitro embryo production techniques (which completely bypass the oviduct) have reinforced this idea. Yet, it became evident that in vitro produced embryos differ markedly from their in vivo counterparts, and these differences are associated with lower pregnancy outcomes and more health issues after birth. Nowadays, researchers consider the oviduct as the most suitable microenvironment for early embryonic development and a substantial effort is made to understand its dynamic, species-specific functions. In this review, we touch on the origin and molecular components of the oviductal fluid in mammals, where recent progress has been made thanks to the wider use of mass spectrometry techniques. Some of the factors and processes known to regulate oviductal secretions, including the embryo itself, as well as ovulation, insemination, endogenous and exogenous hormones, and metabolic and heat stress, are summarized. Special emphasis is laid on farm animals because, owing to the availability of sample material and the economic importance of fertility in livestock husbandry, a large part of the work on this topic has been carried out in domestic animals used for dairy and/or meat production.

Effect of Ionic and Non-Ionic Contrast Media on Aggregation of Red Blood Cells in Vitro

1987 ◽  
Vol 28 (1) ◽  
pp. 87-92 ◽  
Author(s):  
R. Raininko ◽  
S.-L. Ylinen
Keyword(s):  
Contrast Media ◽  
Animal Experiments ◽  
Laboratory Animals ◽  
Animal Blood ◽  
Blood Samples ◽  
Ionic Contrast ◽  
Physiologic Saline ◽  
Meglumine Diatrizoate ◽  
Human Red Cells

Fresh human blood without additives, and contrast medium were mixed and examined immediately by light microscopy in a non-flowing state. Sodium meglumine diatrizoate, meglumine diatrizoate, meglumine iodamide, sodium meglumine ioxaglate, iopromide, iopamidol, iohexol, and metrizamide were tested in concentrations of 300 mg I/ml. Physiologic saline and 5% glucose were used as controls. All media were tested in a randomized order with blood samples from 23 volunteers. No aggregation was detected in physiologic saline, and few rouleaux were found in ionic contrast media. Irregular red cell aggregates were found in all low-osmolal contrast media: in 17 per cent of the specimens in ioxaglate, in 52 per cent in metrizamide, and in 78 to 100 per cent in other non-ionic media. Irregular aggregates were seen in all specimens with glucose. It remains to be demonstrated whether or not the irregular aggregation of human red cells in non-ionic contrast media has clinical significance. Iohexol was also tested with blood samples from several laboratory animals, but in nearly every case no aggregates were found. Results of animal experiments or tests with animal blood seem to be poorly applicable to man.

Epithelial properties of human colonic carcinoma cell line Caco-2: effect of secretagogues

1985 ◽  
Vol 248 (5) ◽  
pp. C410-C418 ◽  
Author(s):  
E. Grasset ◽  
J. Bernabeu ◽  
M. Pinto
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Colonic Carcinoma ◽  
Secretory Cells ◽  
Mucosal Membrane ◽  
Flux Measurements ◽  
Cell Depolarization ◽  
Epithelial Layers

Human colonic carcinoma Caco-2 cells grown in vitro form epithelial layers of highly polarized cells. Unlike colonic adsorptive cells they possess a mucosal membrane with very limited ionic conductance, even after exposure to aldosterone. When grown on filters, Caco-2 cells were sensitive to various secretagogues; these included 10(-5) M dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and 10(-10) M vasoactive intestinal peptide, both of which, added serosally, enhanced the short-circuit current. The same applied to mucosal forskolin. Caco-2 cell sensitivity to serosal epinephrine was lower. Ion substitutions and 22Na-36Cl flux measurements indicated the possibility of secretagogue-dependent chloride secretion. Measurements on cells grown on Petri dishes and exposed to 1 mM DBcAMP for 1 h enabled detection of more profound modifications. Sustained 20-mV cell depolarization and a large reduction in the relative electrical resistance of the mucosal membrane were concomitant with a sizable decrease in 36Cl accumulation. These results suggest that Caco-2 cells, which to some extent resemble colonic crypt cells, possess the cAMP-dependent mucosal chloride conductance characteristic of secretory cells.

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