Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.     

DNA Index in childhood acute lymphoblastic leukaemia: a karyotypic method to validate the flow cytometric measurement

The DNA index (DI) is a prognostic factor in childhood acute lymphoblastic leukemia (ALL). The accuracy of DI measurement is important for treatment stratification: hyperdiploidy with DI ≥ 1.16 is predictive of favorable prognosis whereas hypodiploidy is associated with poor prognosis. The aim of this study was to validate the accuracy of the DI measured by flow cytometry (FCM) by comparison with the karyotype. From samples of 112 childhood ALL, we created a formula to calculate a theoretical DNA index (tDI) based on the blast cell karyotype, taking into account the additional or missing chromosome material of the major clone. FCM DI correlated with tDI calculated from karyotype (R = 0.987) and with modal chromosome number (DI = 0.0202 × Modal NB + 0.0675 and R = 0.984). In three cases a hypodiploid blast cell population was detected by FCM, while only the duplicated clone was identified by the karyotype. The strong correlation between tDI and DI validates the accuracy of FCM quantification, which is technically fast on fresh or frozen samples. If the karyotype is essential to analyze chromosomal abnormalities, FCM provides complementary information in aneuploid ALLs, either by confirming the cytogenetic data or by detecting additional clones not identified when only using cytogenetic data.     

Homozygous alpha6 integrin mutation in junctional epidermolysis bullosa with congenital duodenal atresia

Junctional epidermolysis bullosa with congenital pyloric or duodenal atresia is a distinct variant within this group of autosomal recessive blistering skin diseases. In this study we demonstrate, for the first time, a homozygous mutation in the alpha6 integrin gene (ITGA6) in a family with three affected individuals. For this purpose, we first determined the genomic organization of ITGA6, and placed the gene on chromosome 2q by high resolution radiation hybrid mapping. Heteroduplex analysis of PCR products containing the individual exons of ITGA6, followed by direct nucleotide sequencing, revealed that the proband was homozygous for a G-to-T transversion in the +1 position of intron 12. This mutation, 1856+1G-->T, affects an invariant base of the 5' donor splice site predicting aberrant splicing involving exon 12. The mutation was verified in the proband's DNA by restriction enzyme digestion which also confirmed that the parents were heterozygous carriers of this mutation. Altered expression of alpha6 integrin, which forms a heterodimer with the beta4 subunit at the dermal-epidermal junction, would explain fragility and blistering as a result of minor trauma to the skin.      

Effect of modafinil on plasma melatonin, cortisol and growth hormone rhythms, rectal temperature and performance in healthy subjects during a 36 h sleep deprivation

Modafinil is an alerting substance which has been used successfully to treat narcolepsy. Nothing is known about its effect on hormone secretions. For this purpose, eight healthy young men were enrolled in a double blind trial to test the effects of modafinil on daily plasma melatonin, cortisol and growth hormone (GH) rhythms. Blood was sampled for hormone assays, every hour during the daytime and every 30 min during the nighttime. In addition, rectal temperature and mental performances were determined during the study which comprised 3 sessions, two weeks apart: a 24 h control session including a night with sleep (S1) and two 48 h sessions S2 and S3 with a sleep-deprived night (N1) followed by a recovery night (N2). Modafinil (300 mg2) or placebo were randomly attributed during N1 at 22 h and 8 h.
As expected, performance was improved after modafinil administration and body temperature was maintained or increased. Plasma melatonin and cortisol profiles were similar after modafinil and placebo administration. The levels observed during the recovery and the control nights (N2) displayed no difference. For GH, during both sleep deprived nights, secretion was dramatically reduced compared with the control one, although the number of secretory episodes was unchanged.
These data show that the alerting property of modafinil is not related to an alteration of hormone profiles and suggest that the acute modafinil administration is devoid of short-term side-effects.     

Role of hiatal hernia in delaying acid clearance

The aim of this study was to assess prospectively the relationship of a hiatal hernia to gastro-oesophageal reflux. Ninety five children with symptoms of gastro-oesophageal reflux in whom reflux was demonstrated radiologically were investigated. Oesophageal pH monitoring for 18 hours and endoscopy were performed in all patients. On the basis of radiology, patients were divided into those with hiatal hernia (n = 37) and those without (n = 58). Both groups had the same number of reflux episodes on pH monitoring. However, the median duration of the longest episode was significantly greater in the hiatal hernia group (30 min v 19 min), as was the number of reflux episodes longer than five minutes (5 v 4). The percentage with a pH < 4 just failed to be significantly different (13% v 8%). Hiatal hernia was also found to correlate with the presence of oesophagitis. The presence of a hiatal hernia delays the clearance of acid from the oesophagus and is associated with an increased incidence of oesophagitis.     

The British form of hereditary persistence of fetal hemoglobin results from a single base mutation adjacent to an S1 hypersensitive site 5' to the A gamma globin gene

The G gamma and A gamma genes of an individual homozygous for the British form of A gamma nondeletion hereditary persistence of fetal hemoglobin have been cloned and partially sequenced. The G gamma gene was normal, but the A gamma gene was found to have a single base change (T----C) at -198 bp relative to the cap site. Supercoiled plasmids containing normal gamma-genes or the mutant A gamma-gene displayed an S1-hypersensitive site immediately 5' to the base change.     

Perfusion fixation of rat molar pulp tissue for light microscopy

abstract – Molar pulp tissue of 42 rats was subjected to cardiac perfusion fixation or to immersion fixation with glutaraldehyde and formaldehyde. The importance of variations in pressure and duration of cardiac perfusion was studied. The results indicated that the perfusion method was superior to immersion fixation. Best preservation of the tissues was obtained when perfusion was performed with 1.7% glutaraldehyde for 10–12 min under a pressure of 130 cmH₂O, and leaving the animals for 4 h, without immersion fixation. Also perfusion with formaldehyde resulted in good preservation, provided the solutions were prepared from paraformaldehyde powder. In contrast, solutions made from commercial stock solutions of formaldehyde gave inferior results. For sections stained with hematoxylin-eosin, perfusion with glutaraldehyde was preferable, while perfusion with formaldehyde showed increased sensitivity in demonstration of dentinal tubules stained with alcian blue at pH 3.6. It is suggested that in future studies on rat pulp tissue, perfusion with glutaraldehyde and formaldehyde should replace immersion fixation.     

Diagnosis of intrauterine infection by demonstration of antibody-coated bacteria in the amniotic fluid

Summary. Immunofluorescence examination of amniotic fluid for the occurrence of antibody-coated bacteria was carried out in 72 consecutive women with premature rupture of the fetal membranes to assess this method in the diagnosis of intrauterine infection. Antibody-coated bacteria were demonstrated in 11 women with clinical signs of intrauterine infection, histological amnionitis and heavy growth of one bacterial species. The presence of intrauterine infection was considered possible in another 14 women. One patient had no clinical signs of infection, but positive histological and bacteriological evidence; anti-body-coated bacteria were demonstrated in this patient, but not in the remaining 13 women. In 47 women clinical, histological and bacteriological signs of intrauterine infection were absent, and examinations for antibody-coated bacteria were negative in all of them. It is concluded that the demonstration of antibody-coated bacteria in amniotic fluid indicates the presence of intrauterine infection, and the examination may in some cases be positive before the appearance of clinical signs of infection.