Sister chromatid exchange frequencies in patients with scleroderma and their relatives.

Sister chromatid exchange (SCE) analysis is a sensitive measure of chromosome damage induced by many agents which damage DNA. These agents have been reported to be present in the serum of patients with scleroderma. Chromosome aberrations are thought to be more common in cells of these patients and their relatives than in cells of controls. We have observed that the SCE frequencies in blood lymphocytes of patients with scleroderma and their relatives do not differ from those of controls.     

Decrease in the reaction of DNA repair synthesis in response to nicotinamide or 3-aminobenzamide in ultraviolet irradiated systemic lupus erythematosus lymphocytes

Systemic lupus erythematosus (SLE) lymphocytes failed to accelerate ultraviolet (UV) induced DNA repair synthesis normally observed following inhibition of poly(adenosine diphosphoribose) polymerization. Without inhibitors of poly(adenosine diphosphoribose) polymerase such as nicotinamide or 3-aminobenzamide, DNA repair synthesis following UV irradiation was found to occur normally in the cells of patients. These data suggest that DNA repair is disturbed under special conditions in the lymphocytes of SLE patients.     

Cellular mechanism of DNA-specific antibody synthesis by lymphocytes from systemic lupus erythematosus patients

The cellular mechanism of anti-DNA antibody synthesis in patients with systemic lupus erythematosus (SLE) was studied by DNA-specific solid-phase radioimmunoassay. Anti-DNA antibody synthesis in response to DNA was T-dependent, and the experiments with reconstituted lymphocytes from identical twins discordant for SLE showed that B cells and T cells from SLE patients must cooperate to synthesize anti-DNA antibody. Anti-DNA antibody synthesis by lymphocytes from patients with inactive SLE was enhanced by T4 cells and suppressed by T8 cells in response to DNA. Although T4 cells from patients with active SLE could enhance anti-DNA antibody synthesis by autologous B cells, their T8 cells could not suppress anti-DNA antibody synthesis by autologous B cells. These results indicate that elevated anti-DNA antibody synthesis in response to DNA in patients with active SLE is due to abnormalities of both SLE B cells and SLE T cells. They further indicate that dysfunction of T8 cells from patients with active SLE may, in part, be responsible for deficient regulation of anti-DNA antibody synthesis.     

Induction of sister chromatid exchanges by mitomycin C in lymphocytes of young and old human donors

The kinetics of cell proliferation and sister chromatid exchanges (SCE) were studied in blood lymphocytes treated by mitomycin C (MMC) in young and old human donors. In both the young and old donors MMC inhibited cell proliferation. Spontaneous SCE frequency was not age-dependent. In young donors, MMC induced a dose-related linear increase in SCE frequency. In old donors a significantly reduced SCE rate was induced by 80 ng/ml of MMC, suggesting an altered SCE response to mutagen. As SCE represent some form of DNA repair, the results can be interpreted as an indication of a defective DNA repair in aging human lymphocytes.     

Human DNA repair defects

A number of human genetic diseases have come to be described as being defective in DNA repair. The minimum criterion on which this assignment is based is hypersensitivity to the clastogenic or lethal action of specific DNA damaging agents. In one disease, xeroderma pigmentosum, the molecular evidence for a defect in DNA repair is unequivocal. This condition then acts as a model for dissecting others. For the other diseases the formal evidence for defects in repair is less secure or even lacking. The evidence for repair in each disease is assembled together with any methods that have been used to support the differential diagnosis or for prenatal diagnosis. Attempts to clone human DNA repair genes are in hand and may provide the necessary evidence to decide if all the putative DNA repair defective diseases are genuine.Neoplastic disease and neurological degeneration together with immune defects are frequent clinical features linking this set of diseases, suggesting that effective DNA repair may be important in many aspects of human health.     

Lymphocyte apoptosis in systemic lupus erythematosus: relationships with Fas expression, serum soluble Fas and disease activity.

Lupus specific autoantigens are exposed on apoptotic cells. The increased number of apoptotic lymphocytes reported in systemic lupus erythematosus (SLE) may be attributable to abnormalities of lymphocyte Fas expression or serum soluble Fas. In the present study we analysed the count of circulating apoptotic lymphocytes in SLE patients (n=50), by flow cytometry using Annexin V, compared to rheumatoid arthritis patients (RA, n=20), inflammatory bowel disease patients (IBD, n=20) and normal controls (n=20). Lymphocyte Fas expression and serum soluble Fas were measured and related to numbers of apoptotic lymphocytes. The percentage of apoptotic lymphocytes, determined by Annexin V binding, was significantly increased in peripheral blood of SLE patients (median=4.2%) compared with normal healthy donors (median=1.1%) and IBD patients (median=2. 0%) but not RA (median=3.9%). SLE lymphocyte Fas expression was not significantly different from RA or IBD patients. Serum soluble Fas in SLE patients correlated positively with apoptotic lymphocytes and antibodies to double stranded DNA. This study suggests that increased apoptotic lymphocytes and increased lymphocyte Fas expression may not be specific to SLE. Serum soluble Fas may have a role in the regulation of lymphocyte apoptosis in SLE.     

Decreased Lyn expression and translocation to lipid raft signaling domains in B lymphocytes from patients with systemic lupus erythematosus

OBJECTIVE: B lymphocytes from patients with systemic lupus erythematosus (SLE) are hyperactive and produce anti-double-stranded DNA (anti-dsDNA) autoantibodies. The cause or causes of B cell defects in SLE are unknown. In this study, we determined the level and subcellular distribution of Lyn protein, a key negative regulator of B cell receptor signaling, and assessed whether altered Lyn expression is characteristic of B cells in the setting of SLE. METHODS: Negative selection was used to isolate B lymphocytes from blood. Lipid raft signaling domains were purified from B cells obtained from 62 patients with SLE, 15 patients with rheumatoid arthritis, and 31 healthy controls, by gradient ultracentrifugation. The total Lyn protein level was determined by Western blotting, confocal microscopy, and fluorescein-activated cell sorting (FACS). The distribution of Lyn into lipid raft and nonlipid raft domains was determined by Western blotting and confocal microscopy. Lyn content in B cell subpopulations was determined by FACS. In order to assess B lymphocyte activity, we used (3)H-thymidine incorporation and enzyme-linked immunosorbent assay to measure spontaneous proliferation and IgG and cytokine production by B cells. RESULTS: This study revealed that B lymphocytes from a majority of patients with SLE have a reduced level of Lyn and manifest altered translocation to lipid rafts. An investigation into the mechanisms of Lyn reduction suggested that increased ubiquitination is involved. This was evident from increased ubiquitination of Lyn and translocation of c-Cbl into lipid rafts. Studies of B cell responses showed that altered Lyn expression was associated with heightened spontaneous proliferation, anti-dsDNA autoantibodies, and increased interleukin-10 production. CONCLUSION: This study provides evidence for altered Lyn expression in B cells from a majority of patients with SLE. Altered Lyn expression in SLE may influence the B cell receptor signaling and B cell hyperactivity that are characteristic of the disease.     

Sister chromatid exchanges induced by cancer chemotherapeutic agents in vitro and in vivo: consideration of the hazard of drugs as possible mutagens and carcinogens causing second malignancies

Sister chromatid exchanges (SCEs) induced by chemotherapeutic agents currently used for treating various malignancies were studied in vitro and in vivo. Whether the agents are potentially mutagenic/carcinogenic is discussed on the basis of recent clinicopathological observations on secondary malignancy. In the in vitro studies, the induction of SCEs by various anticancer agents, including three anticancer antibiotics, six alkylating agents, and three antimetabolites, was examined and compared with induction by two known carcinogens, 4-nitroquinoline 1-oxide and N-ethyl-N'-nitronitrosoguanidine. In the in vivo studies, SCE frequencies were examined in the lymphocytes from 74 blood samples drawn from 40 patients with cancer or leukemia at various times after the agents had been administered alone or in combination. The SCE frequencies induced by the anticancer antibiotics were variable because the drugs had different modes of action: generally, the drugs have strong cytotoxicity that must affect the viability of cells. On the other hand, alkylating agents induced significantly high levels of SCE frequencies in vitro; the modes of SCE inducibility were similar to those of the two carcinogens. The fact that cells were viable up to relatively high molar concentrations of the agents and that high SCE levels persisted for a long period suggests that the lesions induced by alkylating agents are long-lived and may therefore be more frequently involved in mutagenesis. This finding may be compatible with the clinical observation that the vast majority of patients with second malignancy are found in the group treated with alkylating agents. Antimetabolites generally showed only a weak SCE induction or none in vivo and in vitro.     

Defects in Notch1 upregulation upon activation of T Cells from patients with systemic lupus erythematosus are related to lupus disease activity

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of autoantibodies and deposition of immune complexes in various organs. T cells play a central role in driving disease progression, and multiple defects in T cells from patients with SLE have been uncovered. Notch signalling is an evolutionarily well-conserved signalling cascade involved in the proliferation, differentiation and apoptosis of T lymphocytes during development and peripheral effector functions. In this study, we investigated the correlation between expression of Notch receptor and the severity of SLE disease. On the contrary to T lymphocytes from healthy controls (n=11), Tlymphocytes from patients with active SLE (n=12) failed to upregulate Notch1 upon in-vitro stimulation as quantified by quantitative real time RT-PCR (P相似文献   

Cellular regulation of anti-nRNP antibody synthesis is different from that of anti-DNA antibody synthesis in patients with systemic lupus erythematosus

Summary The cellular regulation of anti-nuclear ribonucleoprotein (nRNP) antibody synthesis in patients with systemic lupus erythematosus (SLE) was examined and compared with that of anti-double-stranded DNA (dsDNA) antibodies. In vitro antibody production by lymphocytes from SLE patients with antibodies to either dsDNA or nRNP alone was measured using dsDNA-specific and nRNP-specific solid-phase radioimmunoassays (RIA). Lymphocytes of SLE patients with only anti-dsDNA antibodies and normal individuals failed to synthesize anti-nRNP antibody with or without nRNP stimulation. In contrast, lymphocytes from SLE patients with anti-nRNP antibody alone in their sera synthesized in vitro a large amount of anti-nRNP antibody with or without nRNP stimulation. Experiments with reconstituted autologous lymphocytes indicated that B cells and T cells were required for anti-nRNP antibody synthesis. As expected, helper function for antibody synthesis by autologous B cells resided in the T4-cell population and suppressor function in the T8-cell population. T8 cells from SLE patients with anti-nRNP antibody alone suppressed anti-nRNP antibody synthesis by autologous B cells irrespective of clinical activity. This is in contrast to anti-dsDNA antibody production, which was not suppressed by autologous T8 cells. These results indicate that the cellular regulation of anti-nRNP antibody synthesis in SLE is different from that of anti-dsDNA antibody syntheis. Increased anti-nRNP antibody synthesis may be due to increased T4-helper cell function rather than defective T8-suppressor function.     

Increased sensitivity of lymphocytes from patients with systemic autoimmune diseases to DNA alkylation by the methylating carcinogen N-methyl-N-nitrosourea.

Lymphocytes from patients with various diseases associated with autoimmunity showed both impaired capacity to repair O6-methylguanine (a powerful, promutagenic, directly miscoding base lesion) and increased sensitivity to the cytocidal effects of cellular methylation by N-methyl-N-nitrosourea (MNU) compared with normal controls and patients with other disorders. Defective repair of O6-methylguanine was significantly associated with arthritis and myositis in the group with systemic lupus erythematosus (SLE), and increased sensitivity to the toxic action of MNU was associated with the presence of immune complexes and the administration of steroids to patients with Behçet's syndrome. The results indicate that lymphocytes from patients with the autoimmune diseases studied are more susceptible to DNA damage with possible relevance to aetiopathogenesis.     

Repair of UV damaged DNA in systemic lupus erythematosus.

The NZB/NZW hybrid mouse is an animal model of human systemic lupus erythematosus (SLE). Two breeding schemes were devised using NZB, NZW, B/W, and CBA mice, which permit definitive decisions regarding genetic and/or viral origin of the disease. It is proposed that at least two factors must be involved: (1) a genetic abnormality producing hyper-responsiveness to nucleic acid antigens, and (2) a DNA repair defect which results in liberation of DNA and RNA when cells are lethally injured. Evidence is presented for a DNA repair deficit in human SLE lymphocytes following in vitro irradiation with ultraviolet (UV) light. Lymphocytes from adult New Zealand and control mice were found to lack normal amounts of endonuclease necessary for repairing UV damage.     

The evolution of human anti-double-stranded DNA autoantibodies

It has been proposed that the anti-double-stranded DNA (dsDNA) response in patients with systemic lupus erythematosus (SLE) is antigen driven and that DNA or nucleosomes select anti-DNA reactive, somatically mutated B cells. We have used site-directed mutagenesis to systematically revert the somatic mutations of two human anti-dsDNA antibodies from SLE patients to analyze the resulting changes in DNA binding as well as binding to other autoantigens. Our data demonstrate that high-affinity binding to dsDNA and nucleosomes is acquired by somatic replacement mutations in a stepwise manner. Reactivity to surface structures of apoptotic cells is acquired by the same somatic mutations that generate high-affinity dsDNA binding. Importantly, revertant antibodies with germ-line V regions did not show any measurable DNA reactivity. We propose that anti-DNA autoantibodies are generated from nonautoreactive B cells during a normal immune response. B cells may acquire autoreactivity de novo during the process of somatic hypermutation. Nucleosomes, if available in lupus patients because of defects in clearing of apoptotic debris, might subsequently positively select high affinity anti-DNA B cells.     

Impaired release of a soluble natural killer cytotoxic factor in systemic lupus erythematosus

Disease states characterized by abnormalities in immune regulation often demonstrate concomitant abnormalities in cytotoxicity mediated by natural killer (NK) cells. For example, some patients with systemic lupus erythematosus (SLE) have depressed NK activity despite the presence of normal numbers of effector cell:target cell conjugates. This study was designed to determine if defects in NK cell function were directly related to impaired release of a soluble cytotoxic factor. NK activity of peripheral blood mononuclear cells and large granular lymphocytes was measured using 51Cr-labeled K562 target cells in 4-hour release assays. The SLE patients had significantly decreased NK activity relative to normal controls. However, the number of effector cell:target cell conjugates was not different in SLE patients versus control subjects. The release of a soluble natural killer cytotoxic factor (NKCF) by peripheral blood mononuclear cells was measured by cytotoxicity induced in K562 cells. NKCF was released preferentially by suspensions enriched in NK cells (large granular lymphocytes). At a 1:1 dilution, NKCF release was significantly lower in SLE patients than in controls. The release of NKCF correlated well with NK activity. Thus, this study shows that the defect in NK cell activity in SLE patients may be related to an impairment in release of a soluble cytotoxic factor with specificity for NK cell-sensitive targets.     

Single and double stranded DNA binding lymphocytes in the peripheral blood of patients with systemic lupus erythematosus and normal controls.

Specific double (D-DNA) and single stranded (S-DNA) deoxyribonucleic acid binding cells were demonstrated in the peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) by rosette formation with antigen coated red blood cells. The proportion of DNA binding cells in the peripheral blood of patients with SLE was significantly higher than that found in a random population of healthy individuals. Significant numbers of D-and S-DNA binding lymphocytes were found in patients with active disease even when anti-DNA of fluorescent antinuclear antibodies disappeared. The specificity of the DNA binding cells was confirmed by inhibition experiments with D-or S-DNA. Spleen lymphocytes were also examined on one occasion and were found to contain a much higher level of DNA binding lymphocytes than the peripheral blood lymphocytes.     

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.     

Genotoxic effect of albendazole in pediatric patients with hepatic hydatid disease.

OBJECTIVE: Hydatid disease occurs throughout the world and is treated with both surgery and medical administration of albendazole. Some adverse effects of albendazole are known. However, its genotoxic effect on humans has not been reported yet. In this study, we aimed to investigate the genotoxic effect of albendazole on human lymphocytes in vivo. METHODS: The study involved 14 children (eight males and six females) who had undergone operations for hepatic hydatid disease. The ages of the patients ranged from 6 to 13 years. Genotoxicity of albendazole was evaluated as the frequency of sister chromatid exchange (SCE) and micronucleated cells in the patient's lymphocytes. Prior to and after albendazole treatment, blood samples were obtained from these patients for SCE and micronucleus (MN) studies. SCE and MN frequencies of the patients were measured separately before and after albendazole treatment. RESULTS: All patient SCE values increased significantly after albendazole administration (p<0.001). Similarly, MN frequencies in all the patients increased significantly following albendazole treatment (p<0.001). CONCLUSION: This study revealed that both SCE and MN frequencies are higher after albendazole treatment. The results suggest that albendazole may be genotoxic to human lymphocytes in vivo.     

Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and XRCC4 DNA repair genes in systemic lupus erythematosus

Impaired DNA repair efficiency in systemic lupus erythematosus (SLE) patients has been reported in some studies, mainly regarding the repair of oxidative damage, but little is known about repair kinetics towards primarily single-stranded DNA breaks. In the present study, we aimed to investigate: (a) the efficiency of SLE peripheral blood leucocytes in repairing DNA damage induced by ionizing radiation and (b) the association of DNA repair gene (XRCC1 Arg399Gln, XRCC3 Thr241Met and XRCC4 Ile401Thr) polymorphisms in SLE patients, considering the whole group, or stratified sub-groups according to clinical and laboratory features. A total of 163 SLE patients and 125 healthy controls were studied. The kinetics of DNA strand break repair was evaluated by the comet assay, and genotyping for DNA repair genes was performed by PCR-RFLP. Compared with controls, SLE leucocytes exhibited decreased efficiency of DNA repair evaluated at 30 min following irradiation. A significant association with DNA repair gene polymorphisms was not observed for the whole group of SLE patients; however, the XRCC1Arg399Gln polymorphism was associated with the presence of anti-dsDNA antibody. The concomitance of two DNA repair polymorphic sites was associated with the presence of neuropsychiatric manifestations and antiphospholipid antibody syndrome. Taken together, these results indicated that SLE leucocytes repair less efficiently the radiation-induced DNA damage, and DNA repair polymorphic sites may predispose to the development of particular clinical and laboratory features.