Increased sister chromatid exchange in human lymphocyte cultures infected with mycoplasma

The effects of fermenting, poorly arginine-utilizing Mycoplasma fermentans and arginine-utilizing Mycoplasma salivarium on the frequency of sister chromatid exchange (SCE) in cultured human lymphocytes were examined. M. fermentans caused no apparent mitosis inhibition of lymphocytes and the increase in SCE frequency was dependent on the inoculum size of the mycoplasma. An evident increase in SCE frequency was observed in lymphocytes infected with smaller inoculum sizes of M. salivarium whereas there was mitosis inhibition of lymphocytes infected with larger inoculum sizes of the mycoplasma. In lymphocyte cultures infected with M. salivarium, the addition of arginine to the culture medium reduced mitosis inhibition but did not diminish the increase in SCE frequency, indicating that arginine depletion was not involved in causing the induction of SCEs in mycoplasma-infected lymphocytes. With regard to the genetic effectiveness of SCE, these results suggested that mycoplasmas are capable of inducing cytogenetic changes in infected host cells.     

Formaldehyde-induced cytotoxicity and sister-chromatid exchanges in human lymphocyte cultures

The role of the error-prone misrepair pathway in mutagenesis was examined for a series of mutagens in umuC⁺ and umuC36 strains of Escherichia coli. Mutagenesis by ENU, MNU, MNNG and EMS was independent of the umuC⁺ gene function, while mutagenesis by MMS, 4NQO, γ-rays and UV was largely umuC⁺-dependent. Residual mutagenesis following UV-treatment of a umuC^? strain showed the same mutational specificity seen in the umuC⁺ strain. In contrast, the umuC mutation altered specificity substantially in an excision-repair-defective strain that showed a UV-spectrum strikingly different from that seen in an excision-repair-proficient strain. Only one of nine trpE frameshift mutations examined was reverted by UV-light and its reversion was umuC-dependent. In comparison, the dependence of frameshift mutagenesis following ICR 191 treatment was site-specific, suggesting at least two mechanisms of frameshift mutagenesis, one dependent upon misrepair, the other not.The results, together with those of previous reports (Kato and Nakano, 1981; Shinoura et al., 1983), suggest that the umuC⁺ gene exerts it's mutator activity via misrepair of DNA lesions provoking the induction of all types of mutational events, though following UV-irradiation mainly transition events are recovered.     

Increased intrathecal high-avidity anti-tau antibodies in patients with multiple sclerosis

Background

Antibodies against tau protein indicate an interaction between the immune system and the neurocytoskeleton and therefore may reflect axonal injury in multiple sclerosis (MS).

Methodology/Principal Findings

The levels and avidities of anti-tau IgG antibodies were measured using ELISA in paired cerebrospinal fluid (CSF) and serum samples obtained from 49 MS patients and 47 controls. Anti-tau antibodies were significantly elevated intrathecally (p<0.0001) in the MS group. The CSF anti-tau antibody levels were lower in MS patients receiving therapy than those without treatment (p<0.05). The avidities of anti-tau antibodies were higher in the CSF than in the serum (MS group p<0.0001; controls p<0.005). Anti-tau avidities in the CSF were elevated in MS patients in comparison with controls (p<0.05), but not in serum.

Conclusions

MS patients have higher levels of intrathecal anti-tau antibodies. Anti-tau antibodies have different avidities in different compartments with the highest values in the CSF of MS patients.     

Analyses of sister-chromatid exchange and cell-cycle kinetics in mouse T- and B-lymphocytes from peripheral blood cultures

A reliable mouse peripheral blood lymphocyte culture assay has been developed for sister-chromatid exchange analysis. Crucial aspects for optimal mitogenesis include: (1) the addition of 5 X 10(5) leucocytes/ml culture; (2) the use of animals with leucocyte counts from 5 to 7 X 10(6)/ml; and (3) the addition of 6% mouse plasma for the first 24 h of a total 54-h incubation. When 7 micrograms phytohemagglutinin/ml were used to stimulate T-lymphocytes, the mitotic index was 3.4 +/- 0.3%, 28 +/- 2.3% of the metaphases were in first-division, and the SCE frequency/metaphase was 7.3 +/- 0.2 (n = 14 mice). When B-lymphocytes were stimulated with 60 micrograms lipopolysaccharide/ml, the mitotic index was 4.5 +/- 0.3%, 64 +/- 3.3% of the metaphases were in first-division, and the SCE frequency/metaphase was 4.6 +/- 0.1 (n = 7 mice). This culture method consistently yields sufficient numbers of metaphases from both B- and T-lymphocytes for SCE and chromosome-breakage studies.     

Analysis of sister-chromatid exchanges (SCEs) in familial and sporadic multiple sclerosis

The levels of sister-chromatid exchanges (SCEs) were calculated in 17 multiple sclerosis (MS) patients (14 familial and 3 sporadic cases) and 16 healthy controls matched for sex and age. The SCEs were significantly increased for MS patients (p = 0.0002), while there was no statistically significant difference between men and women and between younger and older subjects in both groups. Such factors as familial occurrence and severity of MS, smoking habits, and distribution of lymphocyte subpopulations were discussed. Although there was a significant difference between the MS patients taking medication and the patients taking none (p = 0.038), the latter were still significantly different from the controls (p = 0.035), supporting the fact that the disease itself increases SCEs. Our study, done with 2 doses of BrdU, also shows that the increased SCEs in MS patients are not due to a hypersensitivity to this substance known to be an inducer of SCEs. Thus we suggest that the increased SCEs found are probably disease-related.     

Increased frequency of sister-chromatid exchange induced by dothistromin in CHO cells and human lymphocytes

Dothistromin is a metabolite produced by Dothistromin pini and Cercospora arachidicola. The latter fungus is a pathogen of the peanuts and thus the mycotoxin may be a contaminant of foodstuffs. Dothistromin induces a dose-dependent increase in sister-chromatid exchange frequency in Chinese hamster ovary cells and stimulated human lymphocytes. The increased frequency in human lymphocytes seen with dothistromin is significantly higher among lymphocytes from smokers compared with those from non-smokers.     

Induction of chromosome damage and sister-chromatid exchanges in human lymphocyte cultures by the antitumour antibiotic Neocarzinostatin

Neocarzinostatin (NCS), a chemotherapeutic antibiotic, was investigated for the ability to induce chromosomal aberrations and sister-chromatid exchanges (SCEs) in human lymphocyte cultures. It was observed that the antibiotic causes a cell-cycle delay and reduces the mitotic index. Analysis of the induced chromosomal abnormalities showed that they are mainly chromosome and chromatid breaks; while the frequency of SCEs was increased, the magnitude indicates that NCS cannot be considered a potent inducer of SCEs.     

Calcium concentration in brains from multiple sclerosis patients

Calcium (Ca) concentrations were studied in the central nervous system (CNS) of four patients with definite multiple sclerosis (MS, average age 49 yr) and five controls (average age 68 yr). The Ca concentration was determined by neutron activation analysis in autopsy samples taken from the 26 subanatomical regions of CNS tissues. Although the mean Ca concentration in the 26 CNS regions combined was higher in the four MS patients than in the controls, the content of white matter was lower. Whether or not this significantly lower Ca concentration found in the white matter of MS patients plays an important role in the demyelinating process remains unclear, although that lower concentration seems not be age dependent, but MS specific.     

Oxidative stress in patients with multiple sclerosis

It is well known that brain and nervous system cells are prone to oxidative damage because of their relatively low content of antioxidants, especially enzymatic ones, and of the high levels of both membrane polyunsaturated fatty acids (PUFA) and iron easily released from injured cells. We have investigated the oxidative stress in the blood (plasma, erythrocytes and lymphocytes) of 28 patients affected with multiple sclerosis (MS) and of 30 healthy age matched controls, by performing a multiparameter analysis of non-enzymatic and enzymatic antioxidants--Vitamin E (Vit. E), ubiquinone (UBI), reduced and oxidized glutathione (GSH, GS-SG), superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and fatty acid patterns of phospholipids (PL-FA). PL-FA and Vit. E were assayed by GC-MS; UBI and GSH/GS-SG by HPLC; SOD, GPX and CAT by spectrophotometry. In comparison to controls, patients with MS showed significantly reduced levels of plasma UBI (0.21 +/- 0.10 vs. 0.78 +/- 0.08 mg/ml, p < 0.001), plasma Vit. E (7.4 +/- 2.1 vs. 11.4 +/- 1.8 mg/ml, p < 0.01), lymphocyte UBI (8.1 +/- 4.0 vs. 30.3 +/- 7.2 ng/ml blood, p < 0.001) and erythrocyte GPX (22.6 +/- 5.7 vs. 36.3 +/- 6.4 U/g Hb, p < 0.001). This blood antioxidant deficiency was associated with plasma levels of PL-PUFA--especially C20:3 n-6 and C20:4 n-6--significantly higher than controls. In conclusion, the blood of patients with MS shows the signs of a significant oxidative stress. The possibility of counteracting it by antioxidant administration plus an appropriate diet, might represent a promising way of inhibiting the progression of the disease. Antioxidant supplements should include not only GSH repleting agents, but also Vit. E, ubiquinol, and selenium.     

Orienting reaction in patients with multiple sclerosis

A polygraphic study of the somatic (EMG), autonomic (finger plethysmogram, galvanic skin reaction, respiration, pulse, and EEG (acoustic-evoked potential and EEG-blocking reaction) components of the orienting reaction elicited by an auditory stimulus was performed in 71 patients with multiple sclerosis and in 74 matched normal subjects (control group). The study showed a significantly less intense orienting reaction in patients with multiple sclerosis than in control normal subjects. The severity of this responsiveness disturbance depended on the patients' age at symptom onset, major symptom types on admission, and the relapse frequency. The orienting reaction changes found in patients with multiple sclerosis may be ascribed to the diffuse demyelinating lesions in the nervous system of these patients, which generate, apart from the so polymorphous clinical manifestations of this disease, also important responsiveness disturbances.     

Increased amounts of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TS)

Summary Much greater amounts of small polydisperse circular DNA (spcDNA) have been detected, in cell cultures derived from angiofibromas of six patients with tuberous sclerosis (TS) than in those from the skin of these patients or from the skin of 11 healthy donors. This observation could be confirmed by spreading the DNA of appropriate fractions from CsCl density gradients. The findings suggest the existence of a relationship between the chromosomal instability observed in angiofibroma cultures and the mobilization of spcDNA.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Cytotoxic activity and interferon production by lymphocytes from patients with multiple sclerosis

Peripheral blood lymphocytes from MS patients and from healthy control donors were compared for their ability to mediate spontaneous and antibody-dependent cell-mediated cytotoxicity. They were also compared for their ability to respond to infection with various strains of measles and sSPE viruses with interferon production and enhanced NK activity. Neither SLMC nor ADCC against several different target cells was found to be impaired in the MS population. Furthermore, no defect was detected in the response of patients' lymphocytes to virus challenge in vitro in terms of both activation of NK cells and interferon production. Enhanced NK activity was also induced by an exogenous interferon preparation and by Poly I:C to the same extent in patients and controls.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

Occurrence of sister-chromatid exchange and chromosomal aberration during vitamin A-induced cell differentiation in vitro

Prompted by the recent growth in interest in the mechanisms of vitamin A (VA) action, we studied the effects of VA on the frequency of sister-chromatid exchange (SCE) and chromosome aberration (CA) in a culture system using a fetal Syrian hamster (female) pulmonary epithelial cell line (M3E3/C3). When manipulated by specific culture conditions, the cells in this system could be rendered competent for activation of polycyclic aromatic hydrocarbons. Cells induced to such a state were exposed to 0, 2, 8 and 24 micrograms/ml of VA for 4 days. The average frequency of SCE per metaphase increased from 1.64 at 0 micrograms/ml to 3.44 at 24 micrograms/ml with a moderate degree of dose dependence. In addition, the q-terminal area of X-chromosomes appears to be one of the most specifically vulnerable sites for SCE due to VA. The frequency of CA encompassing triradial, quadriradial, quinqueradial, ring and dicentric chromosomes also increased in a rather sigmoid fashion from 3.6% at 0 micrograms/ml to 14.8% at 24 micrograms/ml. Apart from the frequently demonstrated protective roles or otherwise less often encountered promotional effects of VA in the development of squamous metaplasia, neoplasia, neoplastic transformation or mutation, an alternative interpretation for the current results implies a possible relationship between SCE and CA caused by VA and cell differentiation and/or drug resistance mechanisms.     

Numerical defects in CD8CD28 T-suppressor lymphocyte population in patients with type 1 diabetes mellitus and multiple sclerosis

Type 1 diabetes mellitus (T1D) and multiple sclerosis (MS) are organ-specific autoimmune diseases leading to an attack of auto-aggressive lymphocytes against the pancreatic β-cells and central nervous system, respectively. Using four-colour flow cytometry, T-lymphocyte populations having an important function in autoimmune processes were analyzed. T-regulatory cells (Treg) CD4⁺CD25⁺CD127^low, T-suppressor cells (Ts) CD8⁺CD28⁻, activated helper CD4⁺CD25⁺CD127⁺ and cytotoxic CD8⁺CD25⁺ T-cells and also naive CD4⁺CD45RA⁺ and memory T-cells CD4⁺CD45RO⁺ were compared in the group of patients with T1D (n = 30), MS (n = 31) and in the group of healthy controls (n = 29). Significant differences in Ts cells, activated helper and cytotoxic cells and also memory T-cells were recognized in the group of T1D patients compared to healthy controls. Ts population was significantly lowered in MS patients as well. However, no significant differences were noticed in Treg population. The observed data demonstrate significant differences among patients with T1D and MS in comparison to healthy individuals.     

Comparative study of sister-chromatid exchanges and chromosomal aberrations induced by airborne particulates from an urban and a highly industrialized location in human lymphocyte cultures

Cytogenetic effects induced by extracts of airborne particulates in human lymphocyte cultures were studied with regard to local and seasonal variations. Samples of airborne particulates were collected from an urban and a highly industrialized area in March and October, respectively. All extracts of particulates induced a significant increase of sister-chromatid exchanges (SCE) in a dose-dependent manner. Referring to the induction of sister-chromatid exchanges, local and seasonal differences were observed. Samples from the industrialized area revealed the highest activities. In addition, SCE rates found for March samples were always higher than those for October for both locations. Furthermore, a remarkable, significant induction of chromosomal aberrations occurred with all samples from both locations and sampling periods. Aspects of health risk evaluation for exposed human populations are discussed with respect to the observed cytogenetic effects of airborne particulates in human lymphocyte cultures.     

Manganese,copper, and zinc in cerebrospinal fluid from patients with multiple sclerosis

The concentrations of manganese, copper, and zinc in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and patients with no known neurological disease (control group) were measured. Manganese and copper levels were determined by two different analytical methods: atomic absorption spectrometry (AAS) and high-resolution inductively coupled plasma-mass spectrometry (HR-ICP-MS), whereas zinc levels were determined by HR-ICP-MS only. Manganese levels (mean±SEM) were significantly decreased in the CSF of MS patients (1.07±0.13 μg/L, ICP-MS; 1.08±0.11 μg/L, AAS) compared to the levels in the control group (1.78±0.26 μg/L, ICP-MS; 1.51±0.17 μg/L, AAS). Copper levels were significantly elevated in the CSF of MS patients (10.90±1.11 μg/L; ICP-MS, 11.53±0.83 μg/L, AAS) compared to the levels in the control group (8.67±0.49 μg/L, ICP-MS; 9.10±0.62 μg/L, AAS). There were no significant differences between the CSF zinc levels of MS and control patients. The physiological basis for the differences in manganese and copper concentrations between MS patients and controls is unknown, but could be related to alterations in the manganese-containing enzyme glutamine synthetase and the copper-containing enzyme cytochrome oxidase.