Prevalence of primary blepharospasm in a community of Puglia region, Southern Italy.

The authors ascertained the prevalence of primary blepharospasm (BSP) in a community located in Puglia, a region in Southern Italy, by focusing on neurologists and ophthalmologists. The crude prevalence rates were 133 per million (95% CI, 61--153) for both focal and segmental BSP, 74 per million (95% CI, 24--173) for focal BSP, and 59 per million (95% CI, 16--151) for segmental BSP. Prevalence rate increased with age. Apraxia of eyelid opening and BSP coexisted in one third of cases.

Primary hemifacial spasm and arterial hypertension: a multicenter case-control study.

In a case-control study, the authors found that arterial hypertension occurred more frequently among 115 patients with primary hemifacial spasm than among 115 neurologic controls matched for age (+/-5 years), sex, and referral center. The association was not confounded by education level, smoking history, diabetes, or other diseases (adjusted OR 2.64; 95% CI 1.3 to 5.33, p = 0.007). Hypertension was significantly associated with the outcome in the left-sided group (OR 4.0; 95% CI 1.4 to 11.5), but data concerning patients with right-sided spasm were inconclusive (OR 1.05; 95% CI 0.36 to 3.1). In our sample, hypertension either preceded or followed the onset of hemifacial spasm.

Proton magnetic resonance spectroscopy in primary blepharospasm.

Single-volume proton magnetic resonance spectroscopy, localized to basal ganglia, was carried out in 10 patients with primary blepharospasm (PB) to assess the levels of N-acetyl aspartate (NAA), creatine-phosphocreatine, and choline-containing compounds. NAA was reduced significantly in patients compared with control subjects. This result suggests a striatal neuronal loss in PB.

ICAM 1 expression and fluid phase endocytosis of cultured brain microvascular endothelial cells following exposure to interferon beta-1a and TNFalpha.

We have studied the effect of interferon (IF) beta-1a on the basal and TNFalpha-induced intercellular adhesion molecule 1 (ICAM 1) expression and fluid phase endocytosis (FPE) of horseradish peroxidase in cultured rat brain microvascular endothelial cells. Neither basal ICAM 1 expression nor basal FPE were significantly affected by 24-72 h exposure to 1000 U/ml IFbeta-1a. ICAM 1 induction and FPE enhancement caused by 100 U/ml TNFalpha for 24 h was not influenced by simultaneous administration of 1000 U/ml IFbeta-1a. Treatment of cultures with IFbeta-1a for 48 h followed by 24-h coincubation with TNFalpha (100 U/ml) and IFbeta-1a (1000 U/ml) resulted in significant downregulation of TNFalpha-induced ICAM 1 expression and FPE. Downregulation of TNFalpha-induced ICAM 1 expression was not observed when combined treatment with TNFalpha (100 U/ml) and IFbeta-1a (1000 U/ml) for 24 h was followed by 48 h exposure to IFbeta-1a. We concluded that the blood-brain barrier endothelium may be a target of IFbeta-1a. Further, these in vitro findings may correlate with the results of recent clinical trials indicating that chronic treatment of relapsing remitting multiple sclerosis with IFbeta-1a prevents both clinical exacerbations and the appearance on Magnetic Resonance Imaging of new lesions enhanced by gadolinium which is taken up by increased transendothelial fluid phase vesicular transport.

Sensitivity of lymphocytes from vulcanizers to the in vitro induction of sister chromatid exchanges.

Spontaneous frequencies of sister chromatid exchanges (SCEs) and SCEs induced in vitro by chemicals with different mechanisms of action such as mitomycin C, 4-nitroquinoline oxide, and 3-aminobenzamide were examined in phytohemagglutinin-stimulated peripheral blood lymphocytes from a group of workers in a rubber plant and a control group, both of which had been analyzed for levels of spontaneous SCEs 2 years earlier. An interindividual variability in the induction of SCEs was found after in vitro treatments with the different mutagens, which did not correlate with occupational exposure. This variability in the sensitivity to the induction of SCEs might be correlated to genetic differences among individuals, which have to be taken into account in environmental monitoring programs.

Taurine and ellagic acid: two differently-acting natural antioxidants.

Naturally occurring antimutagenic compounds are extensively analyzed for their capacity to protect cells from induced damage. We selected two agents, taurine and ellagic acid, treated in the literature as antioxidants, but whose activity is insufficiently known. This paper reports on the ability of these agents to act against damage induced by mitomycin-C and hydrogen peroxide in Chinese hamster ovary cells cultivated in vitro. Cytogenetic and cytofluorimetric analyses were performed. Ellagic acid proved to have more than one mechanism of action, probably as a scavenger of oxygen species produced by H2O2 treatment, and as a protector of the DNA double helix from alkylating agent injury. In our experimental conditions, taurine seems able to scavenge oxygen species.

Serum IgG to brain microvascular endothelial cells in multiple sclerosis.

Serum IgG to brain microvascular endothelial cells (BMECs) were assessed in the sera from 50 patients with definite multiple sclerosis, 24 patients with other inflammatory and non-inflammatory neurological diseases and 30 healthy individuals. Standard indirect immunofluorescence on BMEC culture was used as the bioassay system. Positive immunostaining was found in the sera (1:5 to 1:50 dilution) from 0/15 inactive relapsing remitting (RR), 12/16 active RR (p = 0.0001), 1/8 relapsing progressive (RP) and 0/11 primary progressive (PP) patients. No specific binding was detected when sera from neurologic and healthy controls were used. The specificity of the immune reaction for brain endothelium was established by the absence of staining on human umbilical vein endothelial cell and brain pericyte cultures. Gadolinium (Gd)-enhanced magnetic resonance imaging of the brain and spinal cord was performed in 36 MS patients within a 10-day interval from serum collection. Anti-brain endothelium antibodies were found in 9/12 patients with, and in 1/24 patients without Gd-enhanced lesions (p = 0.00002). Regardless of a pathogenetic role in the blood-brain barrier breakdown, serum IgG to BMECs may be a marker of disease activity in RR and RP MS and a factor differentiating RR/RP and PP MS.

Endocytosis of horseradish peroxidase by brain microvascular and umbilical vein endothelial cells in culture: an ultrastructural and morphometric study.

The ability to form tight junctions and the paucity of fluid phase endocytosis showed by brain microvacular endothelial cells (BMECs) make up the structural basis of the blood-brain barrier (BBB). Most studies on cultured BMECs focused on intercellular junctions, whereas endocytosis received lesser attention. We studied endocytosis of horseradish peroxidase in primary and passage 1 and 2 BMEC cultures from rat brain as well as in human umbilical vein endothelial cell (HUVEC) culture. Endocytic activity was also analyzed in passage 1 BMECs treated with lipopolysaccharide (LPS, 1 microg/ml for 4 h), which mimics BBB disruption in bacterial meningoencephalitis. The percent of cytoplasmic area occupied by endocytic profiles (vesicles <70 nm and vacuoles >70 nm) and their mean number per cell were significantly lower in primary and passaged BMEC than in HUVEC cultures. The area and number of endocytic profiles significantly increased in BMECs after exposure to LPS. BMECs cultured under standard conditions may be a suitable model for studying the mechanism of increased fluid phase endocytosis in certain diseases and injury states.

Topoisomerase I activity and cellular response to radiation in Chinese hamster cells.

The aim of the present study was to investigate the synergistic potential of the combination of camptothecin, a specific inhibitor of topoisomerase I, and radiation in the the induction of chromosome aberrations and cell cycle delay in actively proliferating mammalian cells. Synergistic effects of the combined treatments were obtained for induced frequencies of aberrations in exponentially growing Chinese hamster ovary cells. The potentiating effects were more pronounced for aberrations of the exchange type, suggesting that interaction of unrepaired radiation- and camptothecin-induced lesions during replication may be involved in the observed drug-radiation synergism. Cytofluorimetric analysis of cell cycle progression in cells receiving the combined treatments displayed enhanced responses of CHO cells to S- and G2 phase delay induced by the single treatments. To investigate the determinants of the synergistic response, the influence of radiation exposure on the catalytic activity of topoisomerase I was assayed. A decreased plasmid supercoiled DNA relaxation capacity of crude extracts derived from irradiated CHO cells was found which suggests a decrease in the topoisomerase I catalytic activity following irradiation. In addition, a lower sensitivity of the enzyme from irradiated cells to inhibition of topoisomerase I activity by camptothecin was also observed using the same DNA relaxation test.

"Apraxia of eyelid opening" induced by levodopa therapy and apomorphine in atypical parkinsonism (possible progressive supranuclear palsy): a case report.

We report a female patient in whom so-called apraxia of eyelid opening (AEO) developed after the onset of possible progressive supranuclear palsy (National Institute of Neurological Disorders and Stroke criteria) and the introduction of antiparkinsonian medications including levodopa. Although parkinsonian symptoms responded poorly to levodopa, AEO worsened after increasing levodopa dosage and disappeared when levodopa was discontinued. Later, a dose of apomorphine widely accepted for acute tests had no significant effect on limb motor activity but induced AEO. Overall, these observations are grounds for thinking that AEO developing in the course of parkinsonism may be either disease- or drug-related. The possibility of manipulating dopaminergic treatment should always be considered when dealing with AEO associated with parkinsonism.

The production of chromosomal alterations by beta-lapachone, an activator of topoisomerase I.

The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) after exposure to beta-lapachone, an activator of mammalian topoisomerase I, were studied in Chinese hamster cells. A dose-dependent increase in the frequencies of SCE was observed in continuous treatments with beta-lapachone. Chromatid-type aberrations were obtained in cells exposed to beta-lapachone for one cell cycle but also in cells exposed during the G2 phase of the cell cycle, with a marked induction of exchange-type aberrations for both treatment schedules. We therefore propose that activation of topoisomerase I by beta-lapachone results in the production of chromosomal alterations. The cell cycle dependence of beta-lapachone clastogenic effects strongly suggests a mechanism for the formation of chromosomal aberrations after this drug closely resembling the one observed for the topoisomerase I inhibitor, camptothecin.

Interaction between X-ray- and restriction endonuclease-induced lesions in the formation of chromosomal aberrations.

Experiments were performed to analyze the possible interaction between lesions induced by X-rays and restriction endonucleases in the production of chromosome-type exchanges. A stronger interaction was found between X-rays and the AluI-induced 'blunt termini' lesions than between X-rays and the BamHI-induced 'cohesive termini' lesions.

Desmutagenic activity of natural humic acids: inhibition of mitomycin C and maleic hydrazide mutagenicity.

Humic acids are natural components of organic matter widespread in the environment. In spite of the incomplete knowledge about their composition, increasing interest in humic acid activity is justified by their ubiquity. Four different humic acids have been tested in Chinese hamster ovary cells in vitro, both alone and in combination with two well-known mutagens (mitomycin C and maleic hydrazide). Data about sister-chromatid exchanges, mitotic and proliferation indices were collected. Our results, on the whole, indicate: (i) a slight mutagenicity and toxicity of tested humic acids, probably due to chlorination during sample preparation; (ii) a desmutagenic rather than antimutagenic activity of the tested humic acids.

Induction of chromosomal aberrations and SCE by camptothecin, an inhibitor of mammalian topoisomerase I.

The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with camptothecin (CM), an inhibitor of mammalian topoisomerase I. While no chromosome-type aberrations were found in G1-treated cells, instead there was a dose-dependent induction of chromatid-type aberrations. These types of chromosomal alteration were not induced during the treatment itself but during the S phase, as CM is not efficiently removed with the normal washing procedure after treatment.

Chromosomal aberrations induced by restriction endonucleases.

Restriction endonucleases (REs) are able to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells. The G1 phase of the cell cycle seems to be especially sensitive for the induction of chromosomal aberrations by REs. The different capacities of REs to induce chromosomal aberrations are probably correlated with the number of recognition sites in the genome.

Effect of L-ethionine on spontaneous and MMC-induced SCEs in G0 and G1 human lymphocytes under liquid-holding recovery conditions.

L-Ethionine is an ethyl analogue of the amino acid, methionine, well known as a carcinogen but not as a mutagen. Its activity is clearly related to its capacity to hypomethylate DNA and RNA. At a final concentration of 5 mM, L-ethionine completely inhibits DNA synthesis in PHA-stimulated human lymphocytes, probably acting on a methylation reaction critical for the initiation of the S phase. This block can be reversed. Utilizing this capacity of L-ethionine to block cell proliferation, we have studied the influence of G0 and G1 repair of premutational damage (PMD) (equivalent to liquid-holding recovery in bacteria) on spontaneous and MMC-induced SCEs in human lymphocytes. Our results clearly show that L-ethionine in our experimental conditions significantly increases the frequencies of spontaneous and MMC-induced SCEs. In view of the hypomethylating activity of the analogue, we hypothesize that this action at the replication fork level may increase the error-prone ligation of unrepaired lesions, thus influencing the frequency of occurrence of SCEs.

Induction of sister-chromatid exchanges by procarcinogens in metabolically competent Chinese hamster epithelial liver cells.

An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.

Hypersensitivity of lymphoblastoid lines derived from ataxia telangiectasia patients to the induction of chromosomal aberrations by etoposide (VP-16).

Mammalian DNA topoisomerase II represents the cellular target of many antitumor drugs, such as epipodophyllotoxin VP-16 (etoposide). The mechanism by which VP-16 exerts its cytotoxic and antineoplastic actions has not yet been firmly established, although the unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors suggest the involvement of DNA double-strand breaks. In the present study we analyzed the chromosomal sensitivity of lymphoblastoid cell lines derived from ataxia telangiectasia (AT) patients to low concentrations of the drug. Our results indicate that AT derived cells are hypersensitive to the clastogenic activity of VP-16 either when the drug is present for the whole duration of the cell cycle or specifically in the G2 phase, confirming that the induction of DNA double strand breaks, to which AT cells seem typically sensitive, could have an important role in the biological activity of VP-16.

DNA damage and cytotoxicity induced by beta-lapachone: relation to poly(ADP-ribose) polymerase inhibition.

beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5, 6-dione) was previously shown to enhance the lethality of X-rays and radiomimetic agents and its radiosensitizing role in mammalian cells was attributed to a possible interference with topoisomerase I activity. Furthermore, beta-lapachone alone was found to induce chromosomal damage in Chinese hamster ovary (CHO) cells. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its genotoxic action in cultured mammalian cells. Flow cytometry analysis of beta-lapachone-treated CHO cells indicated a selective cytotoxic effect upon S phase of the cell cycle. beta-lapachone produced DNA strand breaks as determined by alkaline elution assay; alkaline elution profiles from treated cells showed a bimodal dose-response pattern, with a threshold dose above which a massive dose-independent DNA degradation was observed. Furthermore, beta-lapachone increased the capacity of crude CHO cellular extracts to unwind supercoiled plasmid DNA, while significantly inhibiting in vitro poly(ADP-ribose) polymerase (PARP). These results suggest that damage induction is probably mediated by the interaction between beta-lapachone and cellular enzymatic function(s), rather than reflecting a direct action on the DNA. We suggest that the inhibition of PARP plays a central role in the complex biological effects induced by beta-lapachone in CHO cells.

Formation of chromatid-type aberrations in G2 stage of the cell cycle.

CHO cells were treated in G1 stage of the cell cycle with chromosome-breaking agents that act in an S-dependent manner. The cells were challenged in G2 stage, before fixation, with various inhibitors of DNA synthesis or repair. Short-wave UV, mitomycin C, decarbomyl mitomycin and 4-nitroquinoline oxide (4NQO) were used as chromosome-breaking agents. The inhibitors of DNA repair or synthesis used were hydroxyurea, aphidicolin and caffeine. Permeabilization of cells followed by a treatment with Neurospora endonuclease (a treatment to convert DNA single-strand breaks into double-strand breaks) did not have any influence on the frequencies of chromatid aberrations induced by the chemicals used, whereas with the inhibitors the extent of potentiation varied depending on the mutagen and the inhibitor used.