Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

Is insurance for children enough? The link between parents'' and children''s health care use revisited

The gutless hydrothermal tubeworm Riftia pachyptila Jones relies mainly on its chemoautotrophic bacterial symbionts to supply nutrients in the form of secreted organic compounds resulting from fixation and incorporation of CO2. In this study, symbionts were purified, tested for viability, and incubated in the presence of labeled CO2. We demonstrated that purified symbionts can be used as a viable alternative to experiments with bacterial cultures. Several organic acids, sugars, and amino acids were labeled, but their fraction of the total label stayed generally constant during the incubation times used. However, increasing fractions of succinate and, to a lesser degree, glutamate were excreted into the incubation medium, indicating that these are probably the main carbon-containing compounds transferred from the symbionts to the host. Glutamate could also account for the transport of nitrogen from the symbionts to the host.     

Increasing breast and cervical cancer screening in low-income women

Active cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome (AIDS) was treated with an intraocular sustained-release ganciclovir implant. A total number of 19 implants were performed in 15 eyes of 9 AIDS patients. The intraocular sustained-release ganciclovir was effective in preventing reactivation of CMV retinitis in 15 of the 19 implants, ineffective in 3, and undetermined in 1. All ineffective cases had been resistant to ganciclovir therapy before the implants. Vision after the therapy was maintained at better than 0.5 except for one eye. There were no serious ocular complications caused by the therapy. Among 5 patients with unilateral CMV retinitis, 2 unaffected eyes developed CMV retinitis during this therapy. In addition, another patient developed presumed CMV infection in other systemic organs. Based on these data, the intraocular sustained-release ganciclovir implant was considered to be useful for the treatment of CMV retinitis in AIDS.     

Serum sertraline and N-desmethylsertraline levels in breast-feeding mother-infant pairs

OBJECTIVE: The authors' goal was to study the serum sertraline levels of breast-feeding mothers and their infants. METHOD: They obtained serum levels of sertraline and N-desmethylsertraline in nine mother-infant pairs. RESULTS: Sertraline levels were very low (less than 2 ng/ml) in seven of the nine infants and low (3 ng/ml) in one. N-Desmethylsertraline levels were also low (6 ng/ml or less) in seven of the nine infants. One infant had a high level of N-desmethylsertraline, and one infant had unusual serum sertraline and N-desmethylsertraline values (half of its mother's levels). All infants were thriving. CONCLUSIONS: Most breast-feeding infants whose mothers were taking sertraline had very low serum levels of both sertraline and N-desmethylsertraline, consistent with published reports. The authors discuss in detail the one infant with unusually high levels.     

Treatment of experimental Staphylococcus epidermidis endophthalmitis with oral trovafloxacin

In this report, a replication-defective herpes simplex virus type 1 (HSV-1) vector has been employed to deliver the Escherichia coli LacZ and HSV thymidine kinase (HSVtk) genes to six human ovarian carcinoma cell lines and the efficacy of gene transfer compared to that of adenoviral vectors in vitro. The transduction efficiency of the LacZ-containing virus TOZ.1 was evaluated qualitatively and quantitatively following infection of the different ovarian cancer cell lines. The therapeutic ability of the HSV-T3 vector, which contains the HSVtk gene, was additionally investigated in comparison to the AdCMVHSVTK. Our results show that HSV-1-mediated gene transfer is quantitatively superior to adenoviral vector in five of the six ovarian cancer cell lines at a 100-fold lower dose in vitro. Our preliminary studies suggest that HSV-1 may be a promising alternative vector for ovarian cancer gene therapy.     

Surgical treatment and outcome in patients with a hepatocellular carcinoma greater than 10 cm in diameter

BACKGROUND: Hepatocellular carcinoma (HCC) over 10 cm in diameter at the time of diagnosis continues to account for a number of patients undergoing hepatic resection. This study evaluated the clinicopathological features and outcome following surgery for large HCC. METHODS: Forty patients with a large HCC (greater than 10 cm) (group 1) resected between 1991 and 1996 were studied retrospectively. They were compared with 245 patients who had smaller HCCs (10 cm or less) (group 2). RESULTS: No patient in group 1 had hepatitis C infection compared with 22.9 per cent in group 2 (P=0.001). Patients in group 1 were significantly younger, had higher alpha-fetoprotein levels (16750 versus 1864 ng/ml; P < 0.001), better liver function, a higher incidence of multiple tumours (27 of 40 versus 42.0 per cent; P=0.003) and venous invasion (35 of 40 versus 52.2 per cent; P < 0.001), and underwent more major resections (37 of 40 versus 26.5 per cent; P < 0.001) than those in group 2. Morbidity and mortality rates and hospital stay were comparable in the two groups. For group 1, the 1-, 3- and 5-year disease-free survival rates were 42, 30 and 28 per cent respectively. Multiple tumours, venous invasion and impaired liver function were factors associated with recurrence. CONCLUSION: Large HCC had specific clinicopathological features. In selected patients, resection is safe and offers the chance of long-term disease-free survival.     

Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery

The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.     

Delta9-tetrahydrocannabinol suppresses macrophage costimulation by decreasing heat-stable antigen expression

Delta9-tetrahydrocannabinol (THC) suppresses several immunologic functions of macrophages. The costimulatory activity of a THC-exposed macrophage hybridoma was investigated by its ability to elicit interleukin-2 secretion by a helper T cell hybridoma activated with immobilized monoclonal anti-CD3 antibody. THC added at culture initiation inhibited the T cell response in a dose-dependent manner. When the macrophages were fixed with paraformaldehyde before culture, THC had no effect on T cell stimulation. However, macrophages, which were preincubated with THC and then fixed, were impaired in delivering costimulatory signals to T cells cultured without THC. The drug's inhibitory effect on macrophage costimulatory activity was reversible. THC exposure also decreased macrophage expression of heat-stable antigen (HSA). Antibody blocking experiments showed that HSA expressed on the macrophages provided an important costimulatory signal, whereas B7-1 and B7-2 molecules had a minor role. Treatment of the macrophages with phosphatidylinositol-specific phospholipase C cleaved HSA, but not the transmembrane B7 molecules, from the cell surface. Similar to THC, enzyme treatment significantly diminished macrophage costimulatory activity, which was also reversible. After drug or enzyme removal, HSA expression returned to the control level by 4 h. Therefore, THC suppresses macrophage costimulatory activity by diminishing cell surface expression of HSA.