Evaluation of an inductively coupled plasma mass spectrometry method for the analysis of sweat chloride and sodium for use in the diagnosis of cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive condition that has an incidence of 1:2500 live births in Northern Europe. Due to the large number of mutations that can result in classical or atypical CF phenotype, the sweat test, which quantifies the amount of chloride and sodium in sweat, is vital in supporting the diagnosis of CF. Patients with CF have raised concentrations of chloride and sodium in their sweat; however, it is the concentration of chloride in sweat which provides the greatest diagnostic sensitivity for CF. METHOD: An inductively coupled plasma mass spectrometry (ICP-MS) method for the analysis of sweat chloride and sodium was evaluated for the routine measurement of sweat collected using the Wescor Macroduct(®) Sweat Collection System. The precision, linearity and agreement with the all laboratories trimmed means (ALTMs) and 'weighed-in' concentrations of sodium and chloride in samples supplied by the UK NEQAS external quality assessment (EQA) Sweat Testing Scheme were assessed. RESULTS: This ICP-MS method for the quantification of chloride and sodium in sweat samples was shown to be accurate, precise and suitable for the routine analysis of sweat chloride and sodium. CONCLUSION: The method performs well and is now used in the routine analysis of sweat in this laboratory.

Genetic diagnosis of PLP gene duplications/deletions in patients with Pelizaeus-Merzbacher disease.

Genetic diagnosis of PLP gene duplications/deletions in patients with Pelizaeus-Merzbacher disease.PMD is an X-linked recessive disorder due to a proteolipid protein (PLP) deficiency. Duplications of PLP gene were shown to be the principle cause of the disorder, accounting for an estimated 50-70% of cases. To define a simple and reliable method for genetic diagnosis of PMD, a group of 42 patients with clinical manifestation of PMD was analyzed by means of real-time quantitative PCR. Parallel fluorescence in situ hybridization (FISH) analysis was performed on the same group of patients. Real-time PCR found seventeen samples had increased gene dosage, whereas FISH detected sixteen duplicated samples. Both methods identified a sample with PLP gene deletion. Our results indicate that real-time PCR is a sensitive and reliable method for the detection of gene duplications/deletions. We further discussed the advantages and limitations of each method in clinical diagnosis of PMD.

HER-2/neu expression in germ cell tumours.

AIMS: To determine the rate of HER-2/neu positivity of germ cell tumours by immunohistochemistry (IHC) and by fluorescence in situ hybridisation (FISH). PATIENTS/METHODS: Ninety six archival, paraffin wax embedded pathology specimens were chosen from four groups of germ cell tumours. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the INFORM assay and confirmed with a centromere 17 probe. RESULTS: Twenty two of 96 specimens overexpressed the HER-2/neu protein when measured by IHC. Only three specimens showed HER-2/neu gene amplification by FISH. There was no correlation between the results obtained by IHC and FISH. CONCLUSIONS: The lack of concordance between IHC and FISH makes it unlikely that overexpression of the HER-2/neu protein in germ cell tumours is of prognostic or therapeutic relevance. Because of the low rate of HER-2/neu gene amplification in germ cell tumours, a clinical trial of trastuzumab treatment in patients with germ cell tumours is not warranted.

Relationship between toxicant transfer kinetic processes and fish oxygen consumption.

Three organic compounds of different hydrophobicity, 1,2,4,5-tetrachlorobenzene (TeCB), 3,4,5,6-tetrachloroguaiacol (TeCG) and 4,6-dichlorobenzenediol (DBD), were chosen as the test chemicals to carry out a series of investigations to look at the relationship between toxicant transfer and fish metabolic rate. A significant correlation was found between the toxicant uptake rate constant (k(1)) and fish oxygen consumption, regardless of fish size and species. This correlation was improved when fish toxicant body load was expressed on a percent body lipid basis. Similarly, there also existed a significant relationship between the toxicant depuration rate constant (k(2)) and fish oxygen uptake for a range of chemicals with different octanol/water partition coefficients (K(ow)).

A physiological model to predict xenobiotic concentration in fish.

A physiological model was developed to estimate fish body toxicant load based on information regarding the chemical exposure regime, fish body weight, lipid content and oxygen uptake. The general model was tested in which an oxygen database (OXYREF) was used to predict fish toxicant body burden. Based on the quantitative analysis, it was shown that the model was reliable and accurate in estimating fish body burden of a number of non-metabolized aquatic toxicants. This modified model possesses some functional reality which enables more realistic predictions, making it useful in the practice of aquatic environmental risk assessment.

Detection of monosomy 7 in bone marrow by fluorescence in situ hybridization. A study of Fanconi anemia patients and review of the literature.

Monosomy 7 is frequently found in the bone marrow of patients with Fanconi anemia (FA), marrow myelodysplasia, or acute myelogenous leukemia and is associated with poor prognosis. In our laboratory, cytogenetic analysis of bone marrow from an FA patient found 2 of 30 cells with monosomy 7, but the results of fluorescence in situ hybridization (FISH) indicated that 83 of 207 cells (40%) had monosomy 7. FISH was then used to analyze two earlier samples from the index case, neither of which had monosomy 7 as determined by standard cytogenetics. The FISH analysis determined that the first sample, taken 19 months earlier, had 8 of 200 cells (4%) with monosomy 7 and the second sample. taken 7 months later, contained 43 of 200 cells (21.5%) with monosomy 7. These results indicate a slow evolution toward monosomy 7 in the patient's bone marrow. Standard metaphase chromosome analysis represents only spontaneously dividing cells, leading us to hypothesize that FISH was detecting monosomy 7 in nondividing cells and that it might be useful in the early detection of abnormal clones. To test this hypothesis, FISH was performed on 13 bone marrow samples from nine patients with FA who did not exhibit monosomy 7 by cytogenetic analysis. Monosomy 7 was detected in 3.44% of nuclei in FA patients and in 3% of nuclei in normal controls. To date, none of these nine FA patients have developed monosomy 7 or leukemia. They are being monitored by standard cytogenetics and by FISH to determine whether monosomy 7 develops and whether it can be detected by FISH prior to its detection by standard cytogenetics. As standard practice, we have adopted FISH analysis for monosomy 7 in all patients with FA.

Nucleolar localization of the microtubule-associated protein tau in neuroblastomas using sense and anti-sense transfection strategies.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in CG neuroblastoma cells which contain nucleolar tau and little cytoplasmic tau. To further establish the presence of tau in the nucleolus of these cells, sense and anti-sense transfection strategies were used. CG neuroblastoma cells were transfected with tau sense cDNA and immunostained with Tau-1. Cytoplasmic Tau-1 staining was greatly increased in CG cells which contain very little endogenous cytoplasmic tau. Nucleolar Tau-1 staining was also increased in certain CG cells indicating an increase in nucleolar tau in a subset of transfected cells. CG cells were also transfected with tau anti-sense cDNA which abolished Tau-1 staining in the nucleolus. These results contribute to a growing body of evidence defining tau as a multifunctional protein found in both the cytoplasm and nucleoli of primate cells.

Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries.

Basement membrane-degrading metalloproteinases (gelatinases) appear necessary for vascular smooth muscle cell migration and proliferation in culture and for intimal migration of cells after balloon injury to the rat carotid artery. We investigated in the present study the secretion of gelatinases from pig carotid artery tissue after balloon injury. Segments of injured artery and segments proximal and distal to the area of injury were removed 3, 7, and 21 days after balloon dilatation. Medial explants from these segments were then cultured for 3 days, and the serum-free conditioned media were subjected to gelatin zymography. Production of 72- and 95-kD gelatinases was quantified by densitometry. Balloon-injured segments secreted significantly more 72- and 95-kD gelatinase than did paired distal segments at all time points. Release of both gelatinase activities was increased at 3 and 7 days relative to segments from uninjured arteries but declined again by 21 days after balloon injury. Similar results were found for gelatinase levels in extracts of arterial tissue. Consistent with the protein secretion data, in situ hybridization demonstrated that the mRNAs for both gelatinases were upregulated after balloon injury. Expression was prominent in medial smooth muscle cells, particularly around foci of necrosis, and in neointimal cells 3 and 7 days after balloon injury; 72-kD gelatinase mRNA persisted after 21 days and was prominent in regrown endothelial cells. The upregulation of gelatinase activity paralleled the time course of smooth muscle cell migration and proliferation in this model. We conclude that increased gelatinase production occurs in response to balloon injury and may play a role in permitting migration and proliferation of vascular smooth muscle cells.

Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells.

Vascular endothelial growth factor (VEGF) expression was examined by immunohistochemistry in 45 prostatic carcinoma specimens and ten benign prostatic tumours (BPH). The majority of carcinoma specimens exhibited cytoplasmic staining for VEGF and showed a trend of increasing expression with dedifferentiation (2p = 0.003). Immunoreactive VEGF was also seen in the prostatic carcinoma cell lines, the order of staining intensity was PC3 > DU145 > LNCaP. Intense granular cytoplasmic staining for VEGF was observed in neuroendocrine-like cells which were seen focally in many of the prostatic specimens. Consecutive sections were incubated with a chromogranin A antibody to confirm the neuroendocrine phenotype of these cells. A significant correlation (P < 0.0001) between the total number of intensely stained VEGF-positive cells and chromogranin A-positive cells was found. A subpopulation of neuroendocrine-like cells also showed intense immunoreactivity for transforming growth factor alpha (TGF-alpha). A correlation was observed (2p = 0.0092) between the intensity of VEGF and TGF-alpha immunostaining in carcinoma cells which were not of neuroendocrine differentiation. The presence of these two angiogenic factors may aid the neovascularisation of carcinomas and their increased expression in tumour-associated neuroendocrine cells may contribute to a more aggressive phenotype.

Tau as a nucleolar protein in human nonneural cells in vitro and in vivo.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in HeLa cells and lymphoblasts as well as in nontransformed fibroblasts and lymphocytes. To confirm the presence of tau in the nuclei of these nonneural cells, immunoblotting analysis was performed on isolated nuclei from lymphoblasts. Several tau bands were noted on the blot of the nuclear extract suggesting the presence of multiple tau isoforms. Tau-1 immunostaining demonstrated variable staining intensities between individual acrocentric chromosomes in all cells tested. In cultured peripheral lymphocytes, these staining patterns were the same from one chromosome spread to the next within an individual. This consistency of Tau-1 staining and its variability among NORs was reminiscent of staining patterns obtained using the silver-NOR procedure. Comparisons of Tau-1 immunostaining with silver staining of chromosome spreads from human lymphocytes demonstrated that Tau-1 did not immunostain all of the NORs that were silver stained. The intensity of Tau-1 fluorescence in nucleoli was further shown to be increased in phytohemagglutinin-stimulated lymphocytes, indicating an upregulation of nuclear tau when cells reentered the cell cycle. These results contribute to a growing body of evidence defining tau as a multifunctional protein that may be involved in ribosomal biogenesis and/or rRNA transcription in the nucleus of all cells as well as microtubule-stabilizing functions in the neuronal cytoplasm.

Endothelial control of vascular smooth muscle proliferation in an organ culture of human saphenous vein.

We have investigated the positive and negative regulation of vascular smooth muscle cell (VSMC) proliferation by proposed endothelium-derived mediators using organ cultures of freshly isolated and surgically prepared human saphenous vein. We observed that: (1) whereas platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are known to stimulate proliferation, agents that minic the action of prostacyclin and nitric oxide (NO), inhibit proliferation; (2) the production of PDGF, bFGF, prostacyclin and NO are endothelium-dependent in veins before culture--PDGF production is induced in VSMC during intima formation; (3) removal of endothelium has a net inhibitory effect on intimal VSMC proliferation; (4) antibodies to bFGF reduced intimal VSMC proliferation in surgically prepared veins. Hence, in this preparation, PDGF, bFGF, prostacyclin and NO are all possible endothelium-dependent regulators of VSMC proliferation. Use of selective inhibitors (as exemplified here by antibodies to bFGF) and reconstitution of endothelium in the organ culture model promise to be valuable to test the roles of these mediators further.

Agent-specific ras oncogene activation in rat thyroid tumours.

Activated ras oncogenes have been detected in a number of rodent tumour model systems (Balmain et al., 1986; Barbacid 1987), in which chemical carcinogens have been the most widely used inducing agents. We have investigated the involvement of activated oncogenes in a single well-characterised tumour model, rat thyroid neoplasia, produced not only by chemical carcinogen (nitrosomethylurea, NMU) and by ionizing radiation but also by elevated trophic stimulation alone. We have indentified a striking specificity in patterns of ras oncogene activation, with exclusively Ha-ras activated in 87% of NMU-induced tumours and exclusively Ki-ras activated in 60% of radiation-induced tumours.

Activated ras oncogenes in human thyroid cancers.

Human thyroid epithelial (follicular) cells give rise to two malignant tumors--"follicular" carcinomas, which metastasize almost exclusively via the bloodstream, and "papillary" carcinomas, which metastasize predominantly via lymphatics (Williams, E. D. In: W. Duncan (ed.), Recent Results in Cancer Research: Thyroid Cancer, pp. 47-55. Berlin: Springer-Verlag, 1980). We have investigated whether this contrast in biological behavior might be associated with different patterns of oncogene activation. DNA transfection analysis of five follicular and ten papillary cancers indeed showed a statistically significant difference in the pattern of genes responsible, activated ras oncogenes being found in 80% of follicular tumors but only 20% of papillary tumors. In addition, in follicular cancers we have found activation of all three ras oncogenes (H-ras, K-ras, and N-ras), the first time that this has been demonstrated in a primary human tumor type (as opposed to cell lines). We suggest therefore that ras activation may be an important determinant of metastatic capability in these epithelial cancers.

The effect of radiation on thyroid C cells.

To study the effects of radiation from 131I on thyroid C cells, new born rats were given 0.5 or 10 muCi 131I and studied at 3 monthly intervals over 2 years. Routine stains, calcitonin immunolocalization and quantitation were used to study follicular and C cells. Both radiation doses led to almost complete disappearance of C cells, with only scattered morphological abnormal surviving cells. After a year, some animals showed focal reappearance of C cells; these isolated cluster were interpreted as clones of cells, derived after a considerable lag period, from the very few surviving C cells which had not been sterilized. This focal regeneration was dose dependent. It is concluded that C cells are much more radiosensitive to radiation from 131I than follicular cells. These observations support the use of 131I therapy in the treatment of post-operative thyroid deposits of medullary carcinoma.