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.     

Comparison of the mutagenic potency of 1,3-butadiene at the hprt locus of T-lymphocytes following inhalation exposure of female B6C3F1 mice and F344 rats

1,3-Butadiene (BD) is an indirect alkylating agent that has greater cancer potency in the mouse than in the rat. The purpose of the present study was to compare the mutagenic potency of BD at the hprt locus of T- lymphocytes of exposed mice and rats and to determine whether mutations induced in this marker gene can be used as a quantitative indicator for species differences in susceptibility to cancer. To this end, experiments were conducted to define the effects of exposure duration and the time elapsed after exposures on the frequency of hprt mutations (Mf) in T-cells from female B6C3F1 mice and F344 rats of similar age (4- 5 weeks) when exposed to BD by inhalation. The accumulation of hprt mutations in T-cells from thymus was assessed in animals necropsied 2 weeks after exposure to 0 or 1250 ppm BD for 1 or 2 weeks, while the time course for the appearance of hprt mutant T-cells (i.e., the phenotypic expression and cell migration) in thymus and spleen was evaluated in animals necropsied at weekly/biweekly intervals up to 10 weeks after exposure for 2 weeks. At necropsy, T-cells were isolated from thymus and spleen and cultured in the presence of IL-2, concanavalin A, and 6-thioguanine (Walker and Skopek, Mutat. Res., 288, 151-162, 1993). BD exposures of 1 and 2 weeks led to mutagenic effects in mouse thymus, with the average Mfs being 3- and 5-fold greater than background values, respectively. In rat thymus, there was only a 1.7- fold increase in Mfs after 2 weeks of BD exposure. In the mutant expression experiment, hprt Mfs in thymus and spleen of both species increased for several weeks post-exposure and then declined. Hprt Mfs in thymus reached maximum levels at 2 weeks post-exposure in mice (Mfs = 11.3 +/- 2.4 x 10(-6)) and at 3 weeks post-exposure in rats (4.9 +/- 1.2 x 10(-6)), while hprt Mfs in spleen reached peak levels at 5 weeks post-exposure in mice (19.7 +/- 1.9 x 10(-6)) and 4 weeks post-exposure in rats (10.1 +/- 1.8 x 10(-6)). Background Mfs for mouse and rat thymus and spleen ranged from 1.6 +/- 0.3 x 10(-6) to 3.0 +/- 1.1 x 10(- 6). Statistical analyses of the hprt Mf data for spleen demonstrated that, under these exposure conditions, the mutagenic potency of BD (represented by the difference in the areas under the phenotypic expression curves of treated versus control animals) was 5-fold greater in mice than in rats. The magnitude of the species differences in mutagenic potency, observed after 2 weeks of BD exposure, resembles the species differences in metabolism more closely than the species differences in cancer potency.      

Role of endogenous secretin in acid-induced inhibition of human gastric function

The role of secretin in the inhibition of gastric acid secretion that occurs during acidification of the gastric lumen was studied in nine healthy men. Gastric acid secretion was stimulated by 500-ml meals of 8% peptone solution, and the pH of the stomach was maintained at 5.5, 2.5, or 2.0 by intragastric titration. The increase in plasma secretin was measured, after extraction, by a new secretin radioimmunoassay. After determining the intravenous dose of secretin required to reproduce plasma secretin concentrations achieved during pH 2.5 and 2.0 meals, similar doses were given during administration of a pH 5.5 peptone meal. The doses of secretin led to plasma secretin concentrations that averaged 3.4 pM, not different from the 3.2 and 3.9 pM concentrations achieved during acidified meals. However, exogenous secretin infusion failed to inhibit acid secretion or gastrin response to peptone, although significant inhibitions occurred in both during peptone meals given at pH 2.5 or 2.0. When secretin infusions were given at fivefold higher rates, plasma gastrin responses again failed to demonstrate significant inhibition. Gastric emptying was inhibited significantly by both acidified peptone meals but only slightly (P = 0.053) during exogenous infusion of physiologic secretin doses. The decrease in acid secretion could be explained by decreased gastrin release, but neither of these findings could be explained by circulating secretin concentrations. These results cast strong doubt on a physiological role of secretin in inhibition of acid secretion in man.     

Acute liver injury induced by weight-loss herbal supplements

We report three cases of patients with acute liver injury induced by weight-loss herbal supplements. One patient took Hydroxycut while the other two took Herbalife supplements. Liver biopsies for all patients demonstrated findings consistent with drug-induced acute liver injury. To our knowledge, we are the first institute to report acute liver injury from both of these two types of weight-loss herbal supplements together as a case series. The series emphasizes the importance of taking a cautious approach when consuming herbal supplements for the purpose of weight loss.     

Evolution of fine needle aspiration cytology in the accurate diagnosis of pancreatic neoplasms

In the era of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), a minimally invasive, safe, and accurate cytologic examination of a variety of intra-abdominal neoplasms has become possible. To assess the efficacy of EUS-FNA for diagnosis of primary pancreatic tumors, a 10-year retrospective review of a consecutive series of patients diagnosed between 1996 and 2005 was undertaken. Comparisons were made between early (1996-2000) and late (2001-2005) periods regarding diagnostic modalities used and the concordance of EUS-FNA cytology with macroscopic tissue histology. Although macroscopic biopsy diagnostic yield did not change over time, yield from EUS-FNA increased from 40 per cent to 95 per cent (P = 0.001). Because of improved accuracy of FNA cytology, only six per cent of tumors required additional macroscopic tissue histology in the late period versus 35 per cent in the early period (P = 0.001). There was 100 per cent concordance between the cytologic and histologic diagnoses in the late period versus only 33 per cent in the early period (P = 0.032). We conclude that (1) the frequency of pathologically diagnosed pancreatic tumors doubled over 10 years, (2) utilization of EUS-FNA significantly increased the accuracy of cytologic diagnosis, and (3) as a result, the need for macroscopic tissue biopsy for diagnosis of pancreatic neoplasms has been obviated.     

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.