Simultaneous determination of planar chlorobiphenyls and polychlorinated dibenzo-p-dioxins and -furans in Dutch milk using isotope dilution and gas chromatography-high-resolution mass spectrometry

A method is described for the simultaneous determination of planar chlorobiphenyls and dioxins in milk using isotope dilution and gas chromatography-high-resolution mass spectrometry (GC-MS). The method is based on gel permeation chromatography, alumina clean-up and carbon chromatography and is highly automated, making a high sample throughput possible. Data on recovery, accuracy and reproducibility of results obtained with quality control samples are presented. Data for both dioxins and planar chlorobiphenyls from the analysis of samples of Dutch milk from several areas in the Netherlands are also presented. Possible interference of the chlorobiphenyls in the determination of the dioxins in the GC-MS method is discussed.     

Video-assisted thoracic surgical resection with the neodymium:yttrium-aluminum-garnet laser

Since January 1991, we have performed 79 video-assisted neodymium: yttrium-aluminum-garnet laser resections for pulmonary nodular or interstitial disease. Pathologic examination demonstrated malignancy in 59 patients (32 primary and 27 metastatic), benign nodules in 11, interstitial processes in seven, and granulomatous disease in two. There were 39 men and 40 women with a mean age of 63.4 +/- 12.5 years. Thirty-nine patients underwent resection with the neodymium:yttrium-aluminum-garnet laser alone and 40 had lesions resected with a combination of laser and endoscopic stapling. Laser excision was performed for lesions deep in the substance of the lung or on its effaced surface; both are locations that make stapling alone difficult. Fifteen of 32 patients with a diagnosis of primary lung malignancy underwent open anatomic resections. Pulmonary reserves of the other 17 patients were inadequate for further resection. Operative time, duration of chest tube placement, length of hospital stay, and complication rate were compared with those for 72 patients undergoing video-assisted thoracic surgical resection of nodules with staplers alone. Although operative time for laser-assisted procedures was longer (p < 0.05), there were no differences in duration of chest tube placement or hospital stay compared with stapled resections. The complication rate for laser-treated cases was not higher than for stapled resections and consisted primarily of air leaks lasting 2 to 7 days. The neodymium:yttrium-aluminum-garnet laser is a safe and precise primary or adjunctive tool for video-assisted thoracic surgical pulmonary resection.     

Manometry of individual segments of the distal esophageal sphincter. Its relation to functional incompetence

The morphology of the arteries in the uterine wall was studied in three multiparous aged mares that had suffered repeated pregnancy failure. The uterine wall arteries exhibited elastosis of the intima or adventitia, or both, resembling "physiological pregnancy sclerosis". In areas affected by elastosis, degeneration of the pre-existing elastic fibres and increased glycosaminoglycans were frequently observed. Newly formed elastic fibres were not evident. Delayed resorption due to disordered metabolic turnover of the elastin was thought to be an important factor in the pathogenesis of the arterial elastosis in the uterine wall.     

Excess expression of uterine ribosomal protein genes P2 and S25 during the "implantation window" in the rat

OBJECTIVE: The present study describes the isolation of "implantation-related" uterine cDNA clones. It further describes their temporal and spatial expression and their identification as the messengers for two ribosomal proteins: RP2 and RS25. METHODS: Libraries of cDNA, representing spayed rats treated with progesterone for 3 consecutive days and with estrogen for either 12 or 36 hours, were screened using homologous and heterologous probes. Two cDNA clones showing differential intensity of the signal were sequenced, the timing of their expression was analyzed by Northern analysis, and their spatial expression was visualized by in situ hybridization. RESULTS: The steady-state level of RP2 mRNA was temporally and spatially controlled by estrogen and progesterone. Whereas estrogen, alone or in combination with progesterone, stimulated this gene, the spatial distribution of the activation was different. Estradiol alone directed RP2 expression to the endometrial-myometrial border, whereas combined treatment increased epithelial staining and directed heavy expression to the stratum vasculare. In normal pregnancy, during the implantation-window period, RP2 was mainly expressed on the mesometrial side, with much less staining on the antimesometrial side. The steady-state levels of the RS25 messenger were elevated by estrogen alone or in combination with progesterone and were confined to the uterine epithelium. RS25 mRNA was evenly distributed throughout the uterine stroma during implantation. CONCLUSION: A developmental pattern of expression of RP2 is reported that corresponds temporally with the primary decidual response but is spatially expressed at the site of the secondary response.     

Ribonuclease L, a 2-5A-dependent enzyme: purification to homogeneity and assays for 2-5A binding and catalytic activity

We have already presented a two-dimensional cell motility assay using a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1 as a motility model of tumour cells of epithelial origin. In this model, L-10 cells showed locomotion as a coherent sheet when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement "cohort migration". Electron and immunoelectron microscopic study of the migrating cell sheets demonstrated localized release from cell-cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex, including beta-catenin. Cell-extracellular matrix (ECM) interactions involved in this TPA-induced cohort migration and their effect on tyrosine phosphorylation of the E-cadherin-catenin complex have now been investigated. L-10 cell cohort migration was almost completely inhibited by addition of Arg-Gly-Asp (RGD) peptide into the medium, and thus RGD dependent. Cohort migration was stimulated on type I and IV collagens, fibronectin (FN)- and laminin-coated substratum, but was inhibited by RGD only on FN-coated surface. By using immunofluorescent techniques, FN was demonstrated preferentially around migrating cells, and a protein synthesis inhibitor, cycloheximide, inhibited the migration by about 75%. FN produced by L-10 cells were found to be mostly EDA+ FN when analysed by RT-PCR. Moreover, anti-FN antibody, but not anti-vitronectin antibody, inhibited the TPA-induced cohort migration almost completely. Thus, it was likely that L-10 cells produced FN themselves and moved on the FN substrate in an RGD-dependent manner. However, stimulation of migration by type I collagen coating and inhibition by RGD treatment did not affect the tyrosine phosphorylation of the E-cadherin-catenin complex induced by TPA, indicating that cell-cell interactions were adjusted to suit cell migration, irrespective of the condition of cell-ECM adhesion, during TPA-induced cohort migration.     

Deep venous thrombosis and neoplastic pathology: our experience in emergencies

Deep vein thrombosis incidence is 1/1000 per year; it is associated with many risk factors which is considered as "thrombophilic states". Its pathogenesis is complex, caused by alterations of hemostasis system. Many studies have established the relation between cancer and subsequent venous thromboembolism, confirming the relationship of neoplastic cell interaction with coagulation system. Forty-seven patients admitted to the hospital from 1987 to 1996 with symptomatic clinically proved deep vein thrombosis were included in a retrospective study. Routine examination at the time of diagnosis of deep vein thrombosis revealed an occult cancer in 8 out of 47 patients; 9 out of 47 patients were admitted in hospital with vein thrombosis and known cancer. The aim of this study is to suggest the best, first treatment of vein thromboembolism in emergency to avoid the dangerous pulmonary embolism complication. The patients affected with deep vein thrombosis and cancer were elderly (over 70 years old, in mean); the neoplasia was of digestive system (8/17) in advanced metastatic stage there was cancer familiarity in 7 out of 47 patients. The high risk of pulmonary embolism associated to deep vein thrombosis suggests the importance of early starting the anticoagulant therapy and placing caval filter.     

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4+ T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/ nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2q) or F1(C57BL/6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further, E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7+ tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in "split" tolerance to that autoantigen.     

Association between carotid lesions evaluated by Doppler color echocardiography and cardiovascular risk factors in the elderly. Preliminary results

BACKGROUND: The valuation of the extracranial carotid by echo color-Doppler takes on an extraordinary importance for the prevention of cerebral ictus at geriatric age. In this "naturalistic" study a population of old people (> 65 yrs) of Cagliari's province was considered in order to: 1) discriminate the lesions of the carotid that are imputed to atherosclerotic disease by anatomic changes of the arterial wall caused by aging; 2) study relations between lesions of the carotid and cardiovascular risk factors. METHODS: The carotids of 50 old subjects were studied by echo color-Doppler and the lesion classified in different classes of severity, according to the hemodynamic standard, comparing them with the presence of the most important cardiovascular risk factors. Hypercholesterolemia was the most frequent risk factor (76%), followed by hypertension (62%), over-weight (54%) and smoking (42%). Moreover a diffused intima-media thickening (IMT) was constant in all the subjects with values > 0.75 mm; athero-sclerotic plaques were even present in 39 subjects which only in 4 cases could be considered at risk of cerebral ischemia. RESULTS: A significant correlation between the severity of the lesions and levels of total cholesterol and LDL cholesterol for the male sex emerged, while for smoking only a trend of correlation has been obtained. CONCLUSIONS: Considering this experience it is suggested that in old subjects the presence of a diffused IMT with values > 0.75 mm must be considered as a marker of aging of the arterial wall of the carotid and not as a cardiovascular risk factor as reported in the literature for the middleaged.     

Different patterns of truncated prion protein fragments correlate with distinct phenotypes in P102L Gerstmann-Str?ussler-Scheinker disease

The clinicopathological phenotype of the Gerstmann-Str?ussler-Scheinker disease (GSS) variant linked to the codon 102 mutation in the prion protein (PrP) gene (GSS P102L) shows a high heterogeneity. This variability also is observed in subjects with the same prion protein gene PRNP haplotype and is independent from the duration of the disease. Immunoblot analysis of brain homogenates from GSS P102L patients showed two major protease-resistant PrP fragments (PrP-res) with molecular masses of approximately 21 and 8 kDa, respectively. The 21-kDa fragment, similar to the PrP-res type 1 described in Creutzfeldt-Jakob disease, was found in five of the seven subjects and correlated with the presence of spongiform degeneration and "synaptic" pattern of PrP deposition whereas the 8-kDa fragment, similar to those described in other variants of GSS, was found in all subjects in brain regions showing PrP-positive multicentric amyloid deposits. These data further indicate that the neuropathology of prion diseases largely depends on the type of PrP-res fragment that forms in vivo. Because the formation of PrP-res fragments of 7-8 kDa with ragged N and C termini is not a feature of Creutzfeldt-Jakob disease or fatal familial insomnia but appears to be shared by most GSS subtypes, it may represent a molecular marker for this disorder.