Applicability and reproducibility of the CPAT-grading system for pancreas allograft thrombosis.

PURPOSE: Although pancreas allograft thrombosis (PAT) incidence has progressively decreased, it remains the most common cause of early graft failure. Currently, there is no consensus on documentation of PAT, which has resulted in a great variability in reporting. The Cambridge Pancreas Allograft Thrombosis (CPAT) grading system has recently been developed for classification of PAT. In this study we aimed to assess the applicability and validate the reproducibility of the CPAT grading system. METHODS: This study is a retrospective cohort study. Selected for this study were all 177 pancreas transplantations performed at our center between January 1 st, 2008 and September 1 st, 2018 were included. RESULTS: A total of 318 Computed Tomography (CT) images was reevaluated according the CPAT system by two local radiologists. Inter-rater agreement expressed in Cohen's kappa was 0.403 for arterial and 0.537 for venous thrombosis. Inter-rater agreement, expressed in the Fleiss' kappa, within clinically relevant thrombosis categories was 0.626 for Grade 2 and 0.781 for Grade 3 venous thrombosis. CONCLUSIONS: Although not perfect, we believe that implementation of the CPAT system would improve current documentation on PAT. However, it is questionable whether identification of a small Grade 1 thrombosis would be relevant in clinical practice. Furthermore, a good quality CT scan, including adequate phasing, is essential to accurately identify potential thrombus and extend after pancreas transplantation.

Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice.

A group of mouse aristaless-related genes has been implicated in functions in the development of the craniofacial skeleton. We have generated an Alx3 mutant allele in which the lacZ coding sequence is inserted in-frame in the Alx3 gene and the sequences encoding the conserved protein domains are deleted. Mice homozygous for this null allele are indistinguishable from wild-type mice. Compound mutants of Alx3 and Alx4, however, show severe craniofacial abnormalities that are absent in Alx4 single mutants. Alx3/Alx4 double mutant newborn mice have cleft nasal regions. Most facial bones and many other neural crest derived skull elements are malformed, truncated or even absent. The craniofacial defects in Alx3/Alx4 double mutant embryos become anatomically manifest around embryonic day 10.5, when the nasal processes appear to be abnormally positioned. This most probably leads to a failure of the medial nasal processes to fuse in the facial midline and subsequently to the split face phenotype. We detected a significant increase in apoptosis localised in the outgrowing frontonasal process in embryonic day 10.0 double mutant embryos, which we propose to be the underlying cause of the subsequent malformations.

Prx1 and Prx2 are upstream regulators of sonic hedgehog and control cell proliferation during mandibular arch morphogenesis.

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. We investigated development of the mandibular arch of Prx1(-/-)Prx2(-/-) mutants to obtain insight into the molecular basis of the lower jaw abnormalities. We observed in mutant embryos a local decrease in proliferation of mandibular arch mesenchyme in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression was strongly reduced, indicative of a function for Prx genes in indirect regulation of SHH: Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopied the lower jaw defects of Prx1(-/-)Prx2(-/-) mutants. In addition, this treatment led to loss of the mandibular incisors. We present a model that describes how loss of Shh expression in Prx1(-/-)Prx2(-/-) mutants leads to abnormal morphogenesis of the mandibular arch.

Targeted inactivation of Hoxb8 affects survival of a spinal ganglion and causes aberrant limb reflexes.

Hoxb8 mutant mice were generated by inserting the lacZ coding sequence in frame with the first exon of Hoxb8. These mice express a fusion protein with a functional beta-galactosidase activity instead of Hoxb8. Mutant embryos were analyzed for anatomical changes. The results indicate that Hoxb8 is not an indispensable regulator of A-P patterning in the forelimb, unlike suggested by our Hoxb8 gain of function experiments (Charité J, DeGraaff W, Shen S, Deschamps J. Cell 1994;78:589-601). The null mutant phenotypic traits include degeneration of the second spinal ganglion (C2), an abnormality opposite to the alteration in the gain of function transgenic mice. Subtle changes in the thoracic part of the vertebral column were observed as well. Adult homozygous mutants exhibit an abnormal clasping reflex of the limbs.

Bmp4 is required for the generation of primordial germ cells in the mouse embryo.

In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of beta-galactosidase activity in Bmp4(lacZneo) embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements.

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs.

Prx1 and Prx2 are closely related paired-class homeobox genes that are expressed in very similar patterns predominantly in mesenchyme. Prx1 loss-of-function mutants show skeletal defects in skull, limbs and vertebral column (Martin, J. F., Bradley, A. and Olson, E. N. (1995) Genes Dev. 9, 1237-1249). We report here that mice in which Prx2 is inactivated by a lacZ insertion had no skeletal defects, whereas Prx1/Prx2 double mutants showed many novel abnormalities in addition to an aggravation of the Prx1 single mutant phenotype. We found defects in external, middle and inner ear, reduction or loss of skull bones, a reduced and sometimes cleft mandible, and limb abnormalities including postaxial polydactyly and bent zeugopods. A single, or no incisor was present in the lower jaw, and ectopic expression of Fgf8 and Pax9 was found medially in the mandibular arch. A novel method to detect &bgr ;-galactosidase activity in hydroxyethylmethacrylate sections allowed detailed analysis of Prx2 expression in affected structures. Our results suggest a role for Prx genes in mediating epitheliomesenchymal interactions in inner ear and lower jaw. In addition, Prx1 and Prx2 are involved in interactions between perichondrium and chondrocytes that regulate their proliferation or differentiation in the bones of the zeugopods.

Intercostal artery: histomorphometric study to assess its suitability as a coronary bypass graft.

To assess potential suitability of the intercostal artery as a conduit in myocardial revascularization a histomorphometric study of the fifth intercostal artery (n = 11) and intercostal arteries other than the fifth (n = 9) was conducted. All arteries were harvested at autopsy in 11 individuals (mean age, 75 years). The length of the intercostal arteries varied from 22 to 35 cm (mean, 29.4 cm). Three combinations of histologic patterns were seen along the course of the intercostal artery: a proximal elastic segment followed by subsequent elastomuscular and muscular segments (n = 3), a proximal elastomuscular segment with the remainder of the artery being muscular (n = 6), and a completely muscular pattern (n = 2). The mean luminal diameter of the fifth intercostal arteries varied from 1.4 +/- 0.3 mm at the origin to 0.9 +/- 0.2 mm at 30 cm (30% to 40% decrease in diameter due to the flaccid state and rigor mortis). The mean intimal thickness at these locations was 54 +/- 38 microns and 25 +/- 16 microns and the mean thickness of the media was 205 +/- 38 microns and 70 +/- 45 microns. The histologic findings, mean luminal diameter, and mean diameter of the intima and the media were similar in the intercostal arteries other than the fifth. Our study demonstrates that the intercostal artery has a relatively thin intima and media, the latter being elastic or elastomuscular in the proximal segment in the majority of the investigated arteries, which are favorable properties with regard to its potential suitability as a coronary bypass conduit.

The circadian control of behavior in the rat affected by the chronic application of methamphetamine.

Chronic application of methamphetamine via the drinking water results in an internal desynchronization of food intake, drinking, and locomotor rhythms in rats. It is discussed whether this may be due to an effect on the central pacemaker system or on more peripheral control centers.