Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.     

Wnt control of stem cells and differentiation in the intestinal epithelium

The intestinal epithelium represents a very attractive experimental model for the study of integrated key cellular processes such as proliferation and differentiation. The tissue is subjected to a rapid and perpetual self-renewal along the crypt-villus axis. Renewal requires division of multipotent stem cells, still to be morphologically identified and isolated, followed by transit amplification, and differentiation of daughter cells into specialized absorptive and secretory cells. Our understanding of the crucial role played by the Wnt/beta-catenin signaling pathway in controlling the fine balance between cell proliferation and differentiation in the gut has been significantly enhanced in recent years. Mutations in some of its components irreversibly lead to carcinogenesis in humans and in mice. Here, we discuss recent advances related to the Wnt/beta-catenin signaling pathway in regulating intestinal stem cells, homeostasis, and cancer. We emphasize how Wnt signaling is able to maintain a stem cell/progenitor phenotype in normal intestinal crypts, and to impose a very similar phenotype onto colorectal adenomas.     

Non-invasive prenatal diagnosis of single-gene disorders from maternal blood

Prenatal diagnosis (PD) is available for pregnancies at risk of monogenic disorders. However, PD requires the use of invasive obstetric techniques for fetal-sample collection and therefore, involves a risk of fetal loss. Circulating fetal DNA in the maternal bloodstream is being used to perform non-invasive prenatal diagnosis (NIPD). NIPD is a challenging discipline because of the biological features of the maternal blood sample. Maternal blood is an unequal mixture of small (and fragmented) amounts of fetal DNA within a wide background of maternal DNA. For this reason, initial NIPD studies have been based on the analysis of specific paternally inherited fetal tracts not present in the maternal genome so as to ensure their fetal origin. Following this strategy, different NIPD studies have been carried out, such as fetal-sex assessment for pregnancies at risk of X-linked disorders, RhD determination, and analysis of single-gene disorders with a paternal origin. The study of the paternal mutation can be used for fetal diagnosis of dominant disorders or to more accurately assess the risk of an affected child in case of recessive diseases. Huntington's disease, cystic fibrosis, or achondroplasia are some examples of diseases studied using NIPD. New technologies are opening NIPD to the analysis of maternally inherited fetal tracts. NIPD of trisomy 21 is the latest study derived from the use of next-generation sequencing (NGS).     

EPITHELIAL CELL MIGRATION IN THE INTESTINE

Despite significant advances in our understanding of the roles of the cytoskeleton and matrix receptors in cell locomotion, derived largely fromin vitrostudies on the movement of epithelial cell sheets and isolated cells, the mechanism of epithelial cell migration in the adult intestine remains an enigma. The primary function of the epithelial cell cytoskeleton seems to be in the maintenance of the apical region of the epithelium facing the gut lumen. There we find the brush border, with its associated enzymes, and the intercellular adhesion complexes that give the epithelium its cohesiveness and its barrier function. Curiously, there is little in the way of an organized cytoskeleton in the basal region of the epithelium adjacent to the basement membrane on which the epithelium is presumed to migrate. In this short review, I focus on what is known about epithelial migration from our understanding of the structure of the epithelium and from studies on wound healing, and indicate some avenues for future study.     

Über die Lymphkapillaren in den Darmzotten von Meerschweinchen und Affe

Zusammenfassung Die zentralen Chylusgefäße in den Zotten des Duodenum und Jejunum von Meerschweinchen und Affe zeigen die Strukturbesonderheiten, die für die Lymphkapillaren des Dünndarms charakteristisch sind. Die Endothelzellen sind durch ihre langen Ausläufer in komplizierter Weise miteinander verzahnt. Sie können an diesen Stellen auseinanderweichen und größeren Chylomikronen den Durchtritt vom extrazellulären Raum in die Lymphkapillaren ermöglichen. Marklose Nervenfasern und vor allem glatte Muskelzellen der Muscularis mucosae treten mit dem Endothel der Lymphkapillaren in Kontakt. Das Zusammenwirken der Endothelverzahnungen mit der Zottenpumpe, die auf den Kontraktionen der glatten Muskelzellen beruht, wird im Hinblick auf den Lymphtransport diskutiert.

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Lymphatic capillaries in the intestine villi of guinea pig and monkey

Summary The central lacteals in the villi of duodenum and jejunum (Macaca mulatta, Cavia cobaya) exhibit the same structural peculiarities as the lymphatic capillaries in the villi of the small intestine in general. The endothelial cells overlap extensively at their margins. At these points they may be separated from each other so enabling larger chylomicra to pass from the extracellular space into the lumen of the lacteal. Nonmyelinated nerve fibres as well as smooth muscle cells of the Muscularis mucosae in particular contact the endothelium of the lymphatic capillaries. The co-operation of the endothelial interdigitations with the villus pump, realized by the contractions of the smooth muscle cells, is discussed as mechanism of the lymph transport.

30. Mai 1972.     

Distribution of 3-hydroxy-3-methylglutaryl-CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.     

Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromium

The cells of the immune system form a strong line of defence against foreign substances. The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro. 5 x 10(6) cells were incubated with 10 or 25 microg ml(-1) of Cr (VI) in the form of K2Cr2O7 at 37 degrees C in the presence of 5% CO2 in air. At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated. Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages. The reduction by splenic and peritoneal macrophages was similar. Total thymocytes reduced 54% of the Cr (VI). Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated. Among the intestinal cells the maximum reduction of 100% (of 10 microg ml(-1)) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells. The reduction in the intestinal loop in situ was 100%. The time taken by each cell type for the peak reduction to Cr (VI) was markedly different. The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium. The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects.     

Intestinal Disaccharidase and Peroxidase Deficiencies during Eimeria nieschulzi Infections in Rats*

SYNOPSIS Experiments were designed to study intestinal pathophysiologic changes associated with coccidial infections in mammalian hosts. Pairs of male Sprague-Dawley rats were killed at various times postinoculation (PI) with 10⁴ or 10⁶ sporulated occysts of Eimeria nieschulzi. The small intestine from each rat was removed, weighed, measured, and divided into thirds. From the middle 11 cm of each third, one cm was fixed for histologic examination. Mucosa was scraped from the remaining 10 cm and was assayed for protein content and for peroxidase, sucrase and trehalase activities. Infection with E. nieschulzi was associated with increased mass of the small bowel. Histologically, crypt depth throughout the small bowel was significantly greater (P≤ 0.005) in infected rats than in non-infected ones on PI days 8 and 16. Villus height did not change drastically during low-dose infections (10⁴ oocysts) and varied during high-dose infections (10⁶ oocysts). As a result of these morphologic changes in the mucosa, crypt/villus ratios were usually significantly greater (P≤ 0.005) in all infected rats throughout the small bowel. In general, increased gut weight and changes in crypt and villus dimensions became evident by PI day 2, were most pronounced at PI day 8, and began to return to control values by PI day 16. Peroxidase, sucrase, and trehalase levels equaled or were slightly higher than in controls on PI day 2, dropped significantly below controls (P≤ 0.05) by PI day 8, and returned to, or exceeded control levels by PI day 16. The intensity of all changes was directly dose-dependent.     

229例流产绒毛染色体核型分析及两种长期培养方法比较

目的:探讨早期自然流产绒毛染色体核型分析在自然流产病因检测中的应用价值,并比较两种长期培养方法的差别。方法:选择孕早期自然流产的孕妇229例,在无菌条件下,从宫腔内取出绒毛,同时或单独经胰酶消化法与切碎贴壁法进行细胞培养,传代之后常规进行G显带,在显微镜下做核型分析。结果:229例流产胎儿绒毛,培养成功206例,成功率为89.96%。异常核型105例,异常率为50.97%,数目异常者101例,占异常核型的96.19%,以16三体最为多见。胰酶消化法的培养成功率及收获时间都显著优于直接贴壁法,差异具有统计学意义(P0.05)。结论:自然流产绒毛染色体核型分析对流产查因具有实用价值。胰酶消化法较切碎贴壁法对流产绒毛长期培养及染色体核型分析更具实用性。     

Cri-du-chat (5p-) syndrome presenting with cerebellar hypoplasia and hypospadias: Prenatal diagnosis and aCGH characterization using uncultured amniocytes

We present prenatal diagnosis of a de novo distal deletion involving 5p(5p15.1 → pter) using uncultured amniocytes in a pregnancy with cerebellar hypoplasia, hypospadias and facial dysmorphisms in the fetus. We discuss the genotype–phenotype correlation and the consequence of haploinsufficiency of CTNND2, SEMA5A, TERT, SRD5A1 and TPPP. We speculate that haploinsufficiency of SRD5A1 and TPPP may be responsible for hypospadias and cerebellar hypoplasia, respectively, in this case.