Does enamelin have pleiotropic effects on organs other than the teeth? Lessons from a phenotyping screen of two enamelin‐mutant mouse lines

We analyzed two mutant mouse lines, ATE1 and ATE2, that carry point mutations in the enamelin gene which result in premature stop codons in exon 8 and exon 7, respectively. Both mutant lines show amelogenesis imperfecta. To establish the effect of mutations within the enamelin gene on different organs, we performed a systematic, standardized phenotypic analysis of both mutant lines in the German Mouse Clinic. In addition to the initially characterized tooth phenotype that is present in both mutant lines, we detected effects of enamelin mutations on bone and energy metabolism, as well as on clinical chemical and hematological parameters. These data raise the hypothesis that enamelin defects have pleiotropic effects on organs other than the teeth.     

Genetically encoded Ca2+-sensor reveals details of porcine endothelial cell activation upon contact with human serum

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca²⁺-messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca²⁺-sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca²⁺-sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca²⁺ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca²⁺-sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.     

Infected bile-induced acute pancreatitis in rabbits

Summary Conclusions Bacteria species commonly found in bile of patients with choledocholithiasis render human bile toxic to the pancreas. The severity of infected bile-induced acute pancreatitis depends on the bacterial species. Infected bile-induced acute pancreatitis turns into a sterile inflammation within 10 d. Background Flow of bile into the pancreatic duct was proposed to cause some forms of gallstone pancreatitis. The development of bile-induced acute pancreatitis at physiologic ductal pressure is known to depend on the bacterial infection of bile. In this study, we investigated the effect of a variety of bacteria species commonly found in bile of patients with choledocholithiasis upon the pancreatic toxicity of human bile. The time-course of pancreatic infection in infected bile-induced acute pancreatitis was also analyzed. Methods In rabbits, the pancreatic duct was kept obstructed throughout the experiment. After 24 h, 50 μL of pancreatic juice was obtained from the congested pancreatic duct and replaced with the same quantity of infected human bile. Bile contained bacteria (10⁷ microorganisms/μL) of species frequently found in choledochal secretions of patients with gallstone disease. Effects on pancreatic morphology were studied after 48 h. In another experiment, the number ofEscherichia coli/mg of pancreatic tissue was determined in a time sequence study following exposure of the rabbit pancreatic duct to 50 μLE. coli-infected bile (10⁷ microorganisms/mL) and temporary (12 h) or permanent duct obstruction. Results Sterile bile was not harmful to the pancreas. Infected bile caused an interstitial-edematous pancreatitis with occasional acinar necrosis. The severity of acute pancreatitis depended on the bacterial species. Following pancreatic duct exposure toE. coli-infected bile, there was complete clearance of the bacteria from the gland with a concomitant interstitial leukocyte infiltration within a period of 2–10 d.     

Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Chickens and pigs are the most important livestock species worldwide. They are not only important sources of food, but also valuable models for evolutionary biology and biomedical science. Pigs share a high anatomical and physiological similarity with humans and are an important species for translational biomedical research, for example, in the areas of cancer, diabetes, neurodegenerative, and cardiovascular diseases (1–3). They also resemble the human pathophenotype more closely than rodents. For example, pig models for familial adenomatous polyposis (FAP) develop polyps in the large intestine as observed in human patients (4), whereas mouse FAP models develop them in the small intestine (5). In contrast to mammals, chickens are phylogenetically distant vertebrates from humans, but they were instrumental in the field of developmental biology due to the easy access to the embryonated egg. They are used for studying neurological and cardiovascular functions (6–8) and provided key findings in B cell development and graft versus host responses (9–11). Genetically modified livestock species also hold great promise for agriculture by offering new approaches for disease control, such as genome-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome or Avian Leucosis Virus (ALV)-resistant chickens (12–15).Due to the lack of fully functional embryonic stem cells, genetic engineering in pigs and chickens has been a laborious, inefficient, and time-consuming procedure (16). The generation of pigs with precise germline modifications required gene targeting in somatic cells followed by somatic cell nuclear transfer. This also is not practical in chickens, where precise alteration of the genome only became possible with recent improvements in the cultivation and manipulation of germline-competent primordial germ cells (PGCs) (17–19). These modified PGCs can be injected into the blood vessel system of stage 13 to 15 (Hamburger−Hamilton [HH]) embryos to produce germline chimeras and, by further breeding, genetically modified chickens.With the advent of synthetic endonucleases such as CRISPR-Cas9 efficiency of targeted germline modification has improved in both species (20–23). It still requires the generation and breeding of new founder lines, which is time consuming in large animals. To circumvent the need for generating germline-modified animals, attempts have been made to carry out genome editing directly in specific organs or tissues (24–27). But this has been hampered by the need to deliver both Cas9 and the required guide RNA (gRNA) and by the limited cargo capacity of viral vectors. To bypass this drawback, Cas9 transgenic mice have been generated, requiring delivery of only the respective gRNAs (28).Here, we describe the generation of both Cas9 transgenic pigs and chickens that ubiquitously express Cas9 endonuclease and provide proof of its function in vitro and in vivo. These animals provide an innovative and efficient model for in vivo genome editing to assess gene function in health and disease.     

In the unloaded lower leg,vibration extrudes venous blood out of the calf muscles probably by direct acceleration and without arterial vasodilation

Purpose

During vibration of the whole unloaded lower leg, effects on capillary blood content and blood oxygenation were measured in the calf muscle. The hypotheses predicted extrusion of venous blood by a tonic reflex contraction and that reactive hyperaemia could be observed after vibration.

Methods

Twelve male subjects sat in front of a vibration platform with their right foot affixed to the platform. In four intervals of 3-min duration vibration was applied with a peak-to-peak displacement of 5 mm at frequencies 15 or 25 Hz, and two foot positions, respectively. Near infrared spectroscopy was used for measuring haemoglobin oxygen saturation (SmO₂) and the concentration of total haemoglobin (tHb) in the medial gastrocnemius muscle.

Results

Within 30 s of vibration SmO₂ increased from 55 ± 1 to 66 ± 1 % (mean ± SE). Within 1.5 min afterwards SmO₂ decreased to a steady state (62 ± 1 %). During the following 3 min of recovery SmO₂ slowly decreased back to base line. THb decreased within the first 30 s of vibration, remained almost constant until the end of vibration, and slowly recovered to baseline afterwards. No significant differences were found for the two vibration frequencies and the two foot positions.

Conclusions

The relaxed and unloaded calf muscles did not respond to vibration with a remarkable reflex contraction. The acceleration by vibration apparently ejected capillary venous blood from the muscle. Subsequent recovery did not match with a reactive hyperaemia indicating that the mere mechanical stress did not cause vasodilation.     

Rapid induction of tumor necrosis factor cytotoxicity in naive splenic T cells by simultaneous CD80 (B7.1) and CD54 (ICAM-1) co-stimulation

The co-expression of B7.1 (CD80) and intercellular adhesion molecule (ICAM)-1 (CD54) on tumor cells can induce tumor immunity and immunological memory. We show here that the non-immunogenic tumor lines Lewis lung carcinoma and B16F10 melanoma, co-transfected with B7.1 and ICAM-1, induced cytotoxic levels of membrane tumor necrosis factor (TNF) on naive syngeneic T cells within 24 h. Membrane TNF expression, primarily on CD4 cells, was responsible for tumor cell lysis by naive spleen cells and could be completely abolished by anti-TNF antiserum. It is suggested that the strong induction of TNF cytotoxicity may be important in the establishment of tumor immunity.