bcl-2 rearrangement in Hodgkin's disease. Results of polymerase chain reaction, flow cytometry, and sequencing on formalin-fixed, paraffin-embedded tissue.

We examined 81 cases of Hodgkin's disease for evidence of the t(14;18) translocation, using the polymerase chain reaction assay on lysates of formalin-fixed, paraffin-embedded tissue. Seven of 74 amplifiable cases (9%) were positive for the translocation, which involves the bcl-2 oncogene and the immunoglobulin heavy chain gene. Two of these cases were sequenced and the breakpoints had the same pattern found in follicular lymphoma. The nuclei from one of the cases were sorted into large and small subpopulations. The t(14;18) signal was more intense in the large nucleus subpopulation, which contained a greater proportion of Reed-Sternberg-like nuclei. These results are consistent with the hypothesis that Reed-Sternberg cells carry the translocation, but they do not exclude the possibility that the translocation is found in cells representing the reactive component of Hodgkin's disease. The results also demonstrate that routinely processed material is suitable for polymerase chain reaction-based analysis of translocations, although the sensitivity is reduced 10- to 100-fold, compared with fresh tissue.     

Cytogenetic characterization of the transgenic Big Blue(R) Rat2 and Big Blue(R) mouse embryonic fibroblast cell lines

The transgenic Big Blue^® Rat2 and Big Blue^® mouse embryonicfibroblast cell lines have been used to complement the transgenicBig Blue® rat and mouse in vivo mutagenesis assays. However,limited information is available regarding the karyology ofthese cell lines. Therefore, we have characterized the ploidy,mitotic index, spontaneous frequencies of chromosome and chromatidaberrations and rate of micronucleus (MN) formation in bothcell lines. We have also characterized the frequency of sisterchromatid exchange (SCE) in transgenic Big Blue^® mouse cells.Big Blue^® Rat2 cells are hyperploid and have extremely highbaseline frequencies of cytogenetic damage. In addition, BigBlue^® Rat2 cells are BrdU-resistant, therefore, SCE frequenciescannot be assessed in these cells. We conclude that Big Blue^®Rat2 cells are not useful for routine cytogenetic toxicologystudies. The transgenic Big Blue^® mouse cell line is polyploidand consistently yields a low mitotic index (1%) in untreatedcells. These mouse cells also exhibited moderately high baselinefrequencies of chromosome and chromatid aberrations, however,baseline frequencies of SCE and of MN were not elevated. TransgenicBig Blue^® mouse embryonic fibroblasts were further studiedfor MN induction following treatment with Nethyl-N-nitrosourea(ENU) for 0.5 h at concentrations of 0.425,0.85 and 1.7 mM.Concentration-dependent increases in MN were observed in thesecells. Thus, while an ENU-induced cytogenetic response usingtransgenic Big Blue^® mouse cells demonstrates that thiscellular model could be used to cytogenetically complement themutagenesis assays, the low mitotic index and the high spontaneousfrequency of chromosome damage confounds its use for routinegenetic toxicology studies. ³To whom correspondence should be addressed. Tel: +1 919 541 3275; Fax: +1 919 541 1460; Email: tindall{at}niehs.nih.gov     

Gluthatione-S-transferase P1 polymorphism I105V in familial and sporadic prostate cancer

Several reports suggest that the glutathione-S-transferase (GST) family of enzymes is involved in a variety of cancers, due to their carcinogen-detoxification properties. A polymorphism in codon 105 of the pi variant (GSTP1 I105V), which affects the enzymatic activity of the enzyme, has been linked to the incidence of cancers from different organs. However, the published data in prostate cancer (PCa) is controversial. Some studies report an association with the GSTP1 I105V polymorphism and sporadic PCa, whereas other studies report no association. Recently, one study showed a positive correlation between the GSTP1 I105V polymorphism and familial PCa in a Japanese population. In the present study, we assessed the correlation of the GSTP1 I105V polymorphism with familial and sporadic PCa in an American population. We analyzed DNA samples from 438 patients with familial PCa, 499 patients with sporadic PCa, and 510 controls. We found no significant association between the GSTP1 I105V polymorphism and familial or sporadic PCa when compared to the control group [odds ratio (OR) =1.0 (0.74-1.37); P=0.58]. Moreover, no association was found after stratification for age of diagnosis, Gleason grade, or lymph node involvement [OR =0.84 (0.65-1.09), P=0.37]. These data indicate that there is no associated risk for sporadic or familial PCa in American families containing the GSTP1 I105V polymorphism.     

Vesicular targeting and the control of ion secretion in epithelial cells: implications for cystic fibrosis.

Non-polarized HT-29 colonic epithelial cells fail to respond to cyclic AMP-generating agonists with increases in plasma membrane anion conduction. Radio-isotopic efflux and patch-clamp experiments revealed that both undifferentiated and differentiated HT-29 colonocytes possess volume- and Ca(2+)-activated Cl- channels. However, only within the apical plasma membranes of the latter were cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels found. CFTR was expressed equally well in both non-polarized and polarized colonocytes. Lack of CFTR-dependent anion conduction was shown to be the result of CFTR retention within a peripheral intracellular compartment. We demonstrate that upon polarization, CFTR moves to the apical plasma membrane via a Brefeldin A (BFA)-sensitive intracellular trafficking pathway.     

Routine antenatal screening for hepatitis B using pooled sera: validation and review of 10 years experience.

AIMS: To validate the sensitivity of universal antenatal screening for hepatitis B surface antigen (HBsAg) by testing pools of 10 sera, and to review 10 years' experience using this method. METHODS: 66,945 antenatal patients were tested between 1986 and 1996 using the pooled method. All sera from 1996 (n = 6050) were retrieved and retrospectively tested individually. An in vitro determination of the effect of pooling on sensitivity was performed by checkerboard neutralisation assay. RESULTS: 26 HBsAg positive women were detected by universal screening over 10 years; 12 had non-European surnames and five had known risk factors for hepatitis B infection. High titre anti-HBs sera in the pool reduced the sensitivity of the HBsAg assay, though the effect was only significant at low levels of HBsAg carriage. CONCLUSIONS: The prevalence of hepatitis B is extremely low in the antenatal population served by Plymouth PHL. Pooling is unlikely to reduce sensitivity enough to lead to significant preventable vertical transmission, and is a cost-effective and valid strategy in areas of low seroprevalence.     

Ethanol-induced conditioned place preference is expressed through a ventral tegmental area dependent mechanism

The authors examined the role of the ventral tegmental area (VTA) and nucleus accumbens (NAc) in the expression of ethanol-induced conditioned place preference (CPP). After cannulas were implanted, male DBA/2J mice underwent an unbiased Pavlovian-conditioning procedure for ethanol-induced CPP. Before preference testing, the mice were injected intra-VTA (Experiments 1 and 3) or intra-NAc (Experiment 2) with the nonselective opioid antagonist methylnaloxonium (0-ng, 375-ng, or 750-ng total infusion; Experiments 1 and 2) or the gamma aminobutyric acid (GABA(B)) agonist baclofen (0-ng, 25-ng, or 50-ng total infusion; Experiment 3). Intra-VTA methylnaloxonium or baclofen decreased ethanol-induced CPP, whereas intra-NAc methylnaloxonium had no effect. These findings indicate that the conditioned rewarding effect of ethanol is expressed through a VTA-dependent mechanism that involves both opioid and GABA(B) receptors.     

Ability of bacteria associated with chronic inflammatory disease to stimulate E-selectin expression and promote neutrophil adhesion.

Porphyromonas gingivalis, Pseudomonas aeruginosa, and Helicobacter pylori have been shown to be associated with adult periodontal disease, chronic lung infections, and peptic ulcers, respectively. The ability of these bacteria to stimulate E-selectin expression and promote neutrophil adhesion, two components necessary for the recruitment of leukocytes in response to infection, was investigated. Little or no stimulation of E-selectin expression was observed with either P. gingivalis or H. pylori when whole cells, lipopolysaccharide (LPS), or cell wall preparations added to human umbilical cord vein endothelial cells were examined. P. aeruginosa was able to induce E-selectin to near-maximal levels; however, it required approximately 100 to 1,000 times more whole cells or LPS than that required by E. coli. Neutrophil-binding assays revealed that LPS and cell wall preparations obtained from these bacteria did not promote endothelial cell adhesiveness by E-selectin-independent mechanisms. In addition, P. gingivalis LPS blocked E-selectin expression by LPS obtained from other bacteria. We propose that lack of E-selectin stimulation and the inability to promote endothelial cell adhesiveness are two additional indications of low biologically reactive LPS. We suggest that this property of LPS may contribute to host tissue colonization. In addition, the ability of P. gingivalis to inhibit E-selectin expression may represent a new virulence factor for this organism.     

Immunoblot analysis of anti-Ureaplasma urealyticum antibody in pregnant women and newborn infants.

The purpose of the present study was to determine the frequency in which antibody reactive to Ureaplasma urealyticum could be detected in a population of pregnant women and newborn infants. Serum samples from a prospective cohort of 80 healthy, U. urealyticum culture-positive and culture-negative pregnant women and a retrospective cohort of 522 infants born at between 25 and 42 weeks of gestation were studied by immunoblot analysis. Cultures of specimens from the lower genital tract were positive for U. urealyticum for 83% of the pregnant women, and serum immunoglobulin G (IgG) antibody which reacted to U. urealyticum was detectable in 93% of the pregnant women. Samples from five women (8%) had increases in the number of anti-U. urealyticum IgG bands over the course of the pregnancy. Samples from four of these five women had corresponding increases in the number of antibody bands present in IgA immunoblots. Six of the 522 samples from newborns or cord blood (1.1%) were positive for anti-U. urealyticum IgA; 5 of these 6 samples were also positive for IgM. The six anti-U. urealyticum IgA-positive infants were distributed as follows; 3 of 67 (4.5%) infants were delivered at 25 to 30 weeks of gestation, 3 of 176 (1.7%) infants were delivered at 31 to 34 weeks of gestation, and 0 of 279 infants were delivered at > or = 35 weeks of gestation. An antibody response to U. urealyticum can be detected in pregnant women and preterm infants and may serve as a marker of infection.     

Microcarrier culture of hepatocytes in whole plasma for use in liver support bioreactors.

Contact activation of plasma clotting may limit the use of some microcarrier types for hepatocyte attachment in a liver assist device. Activation by seven microcarrier types was studied in plasma containing 2-500 units heparin/mL. Clotting was activated by dextran (Cytodex 1 and 2) and collagen-coated (Cytodex 3) microcarriers at 2-25 units/mL (Cytodex 1) and 2-100 units/mL (Cytodex 2 and 3). There was no activation by polystyrene, gelatin, glass or fibronectin-coated polystyrene microcarriers. Compared with culture medium, incubation of HepG2 cells in plasma did not affect cell viability but increased cell number (56.4 versus 65.1 x 10(4) cells; P less than 0.05) and incorporation of [3H]-amino acids into protein (204913 versus 279624 dpm; P less than 0.05). Polystyrene-attached cells demonstrated time-linear protein synthesis, glucose and 7-ethoxycoumarin metabolism. We conclude that polystyrene-attached hepatocytes maintain viability and metabolic activity in plasma and are of potential use in a liver support bioreactor.     

Use of Leu M1 and antiepithelial membrane antigen monoclonal antibodies for diagnosing Hodgkin's disease

Biopsies of 82 patients diagnosed as having Hodgkin's disease were reviewed. Seventeen were reclassified histologically as non-Hodgkin's lymphoma or reactive lymphoid hyperplasia. A substantial number of cases of Hodgkin's disease were negative when stained with Leu M1. Staining for Leu M1 was not found in the cases of non-Hodgkin's lymphoma or reactive lymphoid hyperplasia. With the exception of the lymphocyte predominant nodular subtype of Hodgkin's disease, epithelial membrane antigen staining was seen in a few cases of Hodgkin's disease and non-Hodgkin's lymphomas. This was not a useful discriminating feature.