Molecular cytogenetic parameters in fibroblasts from patients and carriers of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a rare autosomal recessive syndrome. Laboratory investigations have failed to detect any consistent anomaly in cells from XP heterozygotic subjects, although examples of behavior intermediate between normal and XP cells have been reported. To estimate random aneuploidy we applied fluorescence in situ hybridization (FISH) with alpha-satellite probes for chromosomes 8 and 9 and replication pattern for TP53 (p53), ERBB2 (HER-2/neu), and MYCN (N-MYC) loci and for the imprinted SNRPN locus. A significantly higher rate of aneuploidy rate was observed in XP patients and carriers than in controls. The asynchrony pattern was significantly higher in XP carriers and patients with all three coding loci analyzed and significantly lower in XP patients and carriers with the imprinted locus SNRPN than in the control group. Molecular cytogenetic parameters such as random aneuploidy and replication pattern, which are known to reflect chromosomal instability, may be part of the tumorigenesis process. In XP patients and carriers, this genetic instability may represent a potential for developing malignancies.

Werner's syndrome (adult progeria): an affected mother and son presenting with resistant psychosis.

Age-related psychotic conditions may be studied by focusing on the unique group of progeroid syndromes. This report will focus on Werner's syndrome, one of the better defined and studied progeroid syndromes. We applied clinical and histophysiological evaluations to two patients, a mother and son, suffering from Werner's syndrome. Both patients presented with resistant psychosis and evidence of impaired cellular repair mechanisms. Psychiatric morbidity in Werner's syndrome is rarely reported. This syndrome can serve as a possible model for aging-associated development of psychosis.

Clinical, cellular, and molecular features of an Israeli xeroderma pigmentosum family with a frameshift mutation in the XPC gene: sun protection prolongs life.

An Ashkenazi Jewish Israeli family with two children affected with severe xeroderma pigmentosum was investigated. A son, XP12TA, developed skin cancer at 2 y and died at 10 y. A daughter, XP25TA, now 24 y old, was sun protected and began developing skin cancers at 10 y. Their cultured skin fibroblasts showed reductions in post-ultraviolet survival (11% of normal), unscheduled DNA synthesis (10% of normal), global genome DNA repair (15% of normal), and plasmid host cell reactivation (5% of normal). Transcription-coupled DNA repair was normal, however. Northern blot analysis revealed greatly reduced xeroderma pigmentosum complementation group C mRNA. A plasmid host cell reactivation assay assigned the cells to xeroderma pigmentosum complementation group C. Cells from both parents and an unaffected child exhibited normal post-ultraviolet-C survival and normal DNA repair. Sequencing the xeroderma pigmentosum complementation group C cDNA of XP12TA and XP25TA revealed a homozygous deletion of two bases (del AT 669-670) in exon 5 with a new termination site 10 codons downstream that is expected to encode a truncated xeroderma pigmentosum complementation group C protein. Sequence analysis of the xeroderma pigmentosum complementation group C cDNA in cells from the parents found identical heterozygous mutations: one allele carries both the exon 5 frameshift and an exon 15 polymorphism and the other allele carries neither alteration. Cells from the unaffected brother had two normal xeroderma pigmentosum complementation group C alleles. This frameshift mutation in the xeroderma pigmentosum complementation group C gene led to reduced DNA repair with multiple skin cancers and early death. Sun protection delayed the onset of skin cancer and prolonged life in a sibling with the same mutation.

Assessment of DNA damage and repair in human peripheral blood mononuclear cells using a novel DNA unwinding technique.

A newly developed, fast and sensitive microplate assay (Fast Micromethod) was used for the assessment of gamma-radiation-induced DNA damage in peripheral blood mononuclear cells (PBMC) from healthy donors of various ages and from cancer patients undergoing radiotherapy. This assay detects the presence of DNA single-strand breaks and alkali-labile sites by monitoring the rate of DNA unwinding under alkaline conditions using the fluorescent dye PicoGreen, which preferentially binds to double-stranded DNA at high pH (>12.0); it requires only minimal amounts of material (approximately 3 x 10(3) cells/well) and can be performed within 3 hrs. or less. EDTA blood samples were collected from patients not undergoing chemotherapy prior and immediately after irradiation, or were collected from healthy donors and irradiated ex vivo. The results revealed that the amount of DNA strand breaks in PBMC, induced by application of a single dose to patients in the course of radiotherapy treatment, markedly varied between different individuals. To examine the effect of age on DNA damage, the basal levels of DNA damage in PBMC from a total of 30 healthy donors were determined: 10 were 20 to 30 years of age, 10 were 40 to 60 years of age and 10 were >70 years of age. It was found that the mean basal level of DNA damage from donors in the >70-year age group was significantly higher (by 97%) than that of the 20- to 30-year age group and 27% higher than that of the 40- to 60-year age group. Measurements of the level of induced DNA damage in PBMC isolated from blood after 2 Gy irradiation with 60Co gamma-rays revealed no significant differences between donors aged 20-30 and 40-60. However, there was a strong increase (by 2.3- to 2.9-fold) in radiosensitivity in the age group >70. The microplate assay described may be used as a pretherapeutic sensitivity test for the assessment of the individual radiosensitivity of patients prior to radiation therapy.

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing.

The dependence on age of both the basal and the X-radiation-induced levels of apoptosis was examined in human peripheral blood mononuclear cells (PBMC). In the same samples, the base value and the extent of induced DNA single-strand breaks were determined, using a sensitive and fast microplate assay. PBMC were isolated from blood of donors of various age groups (20-30, 40-60 and > 70 years of age) and X-irradiated ex vivo using a 6 MV linear accelerator to give a total exposure of 4 Gy. The mean basal levels of apoptosis in PBMC from donors in the 40-60 year age group and the > 70 year age group were found to be only slightly higher (by 20-10%) compared to that of the 20-30 year age group, whereas the extent of DNA damage strongly and significantly (P < 0.01) increased with age by up to 2-fold. In contrast to the extent of induced DNA damage, which steadily increased in the course of ageing by up to 1.8-fold, there was only a transient increase in the level of induced apoptosis to 1.5-fold in PBMC from X-irradiated blood (4 Gy photons) from donors aged 40-60 followed by a decrease to 0.9-fold in PBMC from old donors (>70), compared to age group 20-30. The results show that X-ray-induced apoptosis and DNA damage in PBMC are not correlated during ageing.

Prevalence of 13 autoantibodies and of the 16/6 and related pathogenic idiotypes in 465 patients with systemic lupus erythematosus and their relationship with disease activity.

With a cross sectional study of 465 consecutive systemic lupus erythematosus (SLE) patients tested for 13 autoantibodies (Aab) and two idiotypes we determined the prevalence of Aab according to disease activity, both general and at particular organ systems. Seventy seven percent of SLE sera had at least one Aab and 56% had it at high titres. Pathogenic idiotypes had a prevalence of less than 10% and 166 sera had Aab to 5 or more antigens and 9 sera had Aab against all 13 antigens tested. Patients with active disease had increased prevalence of Aab to DNP, ssDNA, ENA, mitochondria and histones when considered at 5 s.d. above the mean of normal controls. The higher positivity of Aab in patients with active disease was confirmed in logistic regression analysis adjusted by age, disease duration, and intensity of treatment. A trend was observed of increased prevalence and titres of Aab from inactive disease without treatment, to inactive disease but still being treated, to active disease. Only 22% of patients with active disease had no Aab and the higher the number of Aab the higher the frequency of active disease. Patients with active arthritis, and to a lesser degree those with active mucocutaneous involvement, had higher prevalence and titres of most autoantibodies than patients with disease activity at other organ systems. Active renal disease associated only with anti-dsDNA, whereas active CNS disease associated with anti-mitochondrial Aab. Our findings support the vision of SLE as an immune dysregulation leading to polyclonal B cell activation with resulting production of multiple Aab. Their profiles seem influenced by genetical, hormonal and environmental factors and, in turn, they contribute to the clinical picture in each patient. Disease activity influences the presence of some, but not all, Aab and some of them may remain present in some patients, even in remission.

The effect of chlorpromazine and haloperidol on DNA transcription.

The ability of human cells to repair DNA damage can be indirectly assessed by measuring transcriptional activity relating to active genes, a process referred to as RNA synthesis. This study was carried out to investigate the effects of chlorpromazine and haloperidol on the transcriptional activity of actively transcribed genes as an expression of DNA damage and repair. Three cultured human fibroblast lines were used: two were "normal" in previous RNA recovery testings and one was abnormally sensitive to UV irradiation. In the "normal" line, recovery of RNA synthesis occurred within 1 hour of UV after exposure to three concentrations of chlorpromazine (125, 250 and 500 ng/ml) and haloperidol (5, 10 and 20 ng/ml). Following treatment with the same concentrations of chlorpromazine and haloperidol, the UV-sensitive cell line showed markedly depressed recovery of RNA synthesis at 1 and 4 hours. Complete recovery was not reached even after 24 hours. Our results suggest that neuroleptics widely used in clinical practice adversely affect cell lines that are sensitive to DNA-damaging agents.

Anticardiolipin antibodies are elevated in drug-free, multiply affected families with schizophrenia.

The objective of this study was to measure anticardiolipin antibodies in patients and healthy relatives in multicase families with schizophrenia. Twenty-eight (28) multicase families with schizophrenia were examined. One hundred three drug-free patients and 66 first-degree relatives consented to evaluation by DSM-III-R criteria. Criteria for patient definition included the following: age > or = 16, a confirmed hospital diagnosis of schizophrenia, knowledge of biological parents, and consent to participate. Additional data were drawn from family history and medical records. Serum samples were tested separately for IgG and IgM anticardiolipin by enzyme-linked immunosorbent assay (ELISA) and designated positive/negative by comparison to the reactivity of an age-matched control group. IgG anticardiolipin antibodies were significantly more common in both patients and relatives compared to controls. IgM anticardiolipin antibodies were significantly more common in patients. In 75% of families at least one member was anticardiolipin positive and this positivity correlated with patient positivity. The relevance of anticardiolipin antibodies in both patients and healthy relatives of some multicase families to the pathogenesis of schizophrenia is discussed.

Increased anti-Sm antibodies in schizophrenic patients and their families.

1. Autoantibodies in the Sm complex have become a useful serologic aid in the diagnosis of systemic lupus erythematosus (SLE) and have rarely been observed in other diseases. 2. A subset of SLE patients have a variety of psychiatric abnormalities, including schizophrenia. 3. The authors have recently observed that schizophrenic patients have a high incidence of autoantibodies suggesting that autoimmune phenomena may play a role in the pathogenesis of this disease. 4. In the present study the authors investigated multicase families with schizophrenia for the presence of anti-Sm antibodies and showed that these autoantibodies are elevated both in patients and in their healthy relatives. 5. An autoimmune process may be involved in the pathology of schizophrenia.

DNA repair and recovery of RNA synthesis in uremic patients.

A high frequency of cancer appears among uremic patients. As depressed DNA repair ability is thought to be one of the causes for malignancy in cancer prone diseases, the present study was undertaken to examine DNA repair in uremic patients. Unscheduled DNA repair synthesis in peripheral lymphocytes was measured after both ultraviolet (UV) and gamma irradiations. In hemodialysis (HD) patients the repairs were normal, but in chronic renal failure (CRF) patients not yet on dialysis treatment, both UV- and gamma-induced DNA repair abilities were depressed to about 60% of the control. Recovery of RNA synthesis after UV irradiation followed the same pattern: it was reduced in CRF but normal in HD cells. When CRF lymphocytes were incubated in normal plasma, the UV-stimulated DNA repair improved to a nearly normal level, whereas incubation of normal cells in CRF plasma depressed their repair capacity to 70% of the initial level. These results suggest that a plasmatic substance such as the carcinogenic heterocyclic amines may be involved in the impairment of DNA repair in chronic renal failure.

Autoantibodies to DNA in multicase families with schizophrenia.

In an attempt to define the autoimmune status of members of multicase families with schizophrenia, sera of both patients and healthy relatives from 28 such cases were tested for antinuclear antibodies, anti-double-stranded DNA, and anti-single-stranded DNA autoantibodies. These autoantibodies were significantly more frequent in both schizophrenic patients and healthy relatives than in normal subjects. Immunoglobulin (Ig) M anti-DNA antibodies were more common in patients, whereas in healthy relatives, IgG anti-DNA antibodies were more common. No significant differences were found between schizophrenic patients and their healthy relatives. The data indicate that an autoimmune process may be involved in the etiology of a subset of patients with schizophrenia.

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.

Diversity and pattern of inheritance of autoantibodies in families with multiple cases of systemic lupus erythematosus.

The pattern of inheritance of autoantibodies in eight families chosen from a pool of 110 families of patients with systemic lupus erythematosus (SLE) is described. In all the eight families at least two members were already affected by SLE. In total, 19 patients and 43 first degree relatives were examined. The inheritance of a large set of antinuclear antibodies (for example, DNA, Sm, RNP, Ro, La, histones) and 16/6 idiotype seemed to be related to some unknown genetic factors but not related to HLA. The presence of numerous antinuclear autoantibodies in the serum of a subject was not necessarily associated with overt disease. The incidence of the 16/6 idiotype among patients and their relatives was low. It is not yet clear whether the 'autoantibody burden' is greater in families with multiple cases of SLE than in families with single cases.

The effects of incubation temperature and coating procedure on the measurement of antibodies to cardiolipin.

Many laboratories have established ELISAs for the the routine detection of anti-cardiolipin antibodies (ACA). Earlier studies had indicated that assay incubation at 37 degrees C may interfere with the antigen binding capacity of these antibodies. We have reexamined this phenomenon by comparing ACA titers obtained when incubations are performed at either 37 degrees C or at room temperature (RT). In addition, the effect of coating antigen in aqueous or organic solution was compared. The sera tested included a set of recognized ACA standards and samples from 19 patients with SLE, two with primary anti-phospholipid syndrome, 71 patients with a variety of autoimmune and non-autoimmune disorders and 210 blood bank controls. The results show that while some sera do perform better under either incubation temperature there was no correlation between ACA titers and incubation temperature on a population basis either for IgG or IgM isotypes. This was seen both for positive standards and patient sera. For IgG ACA a similar phenomenon was seen if the microplates were coated with cardiolipin either in sodium carbonate or ethanol. For IgM ACA there was a significant increase in ACA titers at RT when cardiolipin was coated in ethanol. The data suggest that for most sera neither the antigen coating medium nor the assay incubation temperature are important variables in the determination of IgG ACA. Factors contributing to the influence of either variable in individual sera could not be identified.

Antinuclear autoantibodies in healthy nonpregnant and pregnant women and their offspring.

Antinuclear autoantibodies have previously been detected in sera of healthy women although less frequently than in sera of women with autoimmune disorders. The effect of pregnancy on antinuclear autoantibody production in healthy women is as yet debatable. We present four studies in which, by employing the ELISA method, we evaluated the presence of six antinuclear autoantibodies (anti-ds DNA, anti-ss DNA, anti-poly(I), anti-cardiolipin, anti-Sm, and anti-RNP) in the sera of more than 1,000 healthy pregnant and nonpregnant women, including 196 pairs of matched maternal and cord blood sera. In all four studies healthy pregnant women did not demonstrate significantly higher prevalence rates of various serum antinuclear autoantibodies as compared to healthy non-pregnant women. All detected autoantibodies were of the IgM isotype. In only one infant (born to a healthy seronegative mother) was an autoantibody (IgM anti-ss DNA) detected. This may indicate that in certain circumstances the fetus is capable of self-production of autoantibodies.

Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes.

During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with epidermal growth factor receptors.

Anti-Sm-RNP activity in sera of patients with rheumatic and autoimmune diseases.

The sera of various rheumatic and autoimmune diseases were examined for the presence of anti-RNP/Sm activity. An enzyme-linked immunosorbent assay (ELISA) was employed. Anti-RNP Ab's were detected in 18%, 20%, 28%, 16% of the sera of SLE, myasthenia gravis (MG), rheumatoid arthritis (RA) and thyroid diseases respectively. The anti-RNP Ab's belonged to the IgG and IgM isotypes. Most of the IgG anti-Sm antibodies were detected in SLE sera, but they were found also in two sera of MG and in one sera of RA patients. IgM anti-Sm antibodies were not found in SLE sera, but they were detected in low titer in MG, RA and autoimmune thyroid diseases. The activity against RNP and/or Sm was further confirmed by employing immunoblotting assays. In none of the patients, except those with SLE, was any clinical manifestation of SLE noted. The mere presence of anti-Sm antibodies of the IgG isotypes is not sufficient for the development of SLE, however, its presence is highly specific for SLE.

Autoantibody production by patients infected with Leishmania.

Sera from 29 patients with visceral leishmaniasis and 14 patients with cutaneous leishmaniasis were tested against a panel of nine nuclear antigens employing an enzyme-linked immunosorbent assay (ELISA). Anti- Sm, RNP, SS-A and SS-B antibodies were present in high titres in 83, 86, 36 and 73 per cent of the patients with visceral leishmaniasis and in 7, 14, 25 and 25 per cent of the patients with cutaneous leishmaniasis. One serum from a patient with visceral leishmaniasis which reacted strongly with Sm, RNP, SS-A and SS-B was examined by immunoblotting on extractable nuclear antigen from Hela cells. This serum binds to nine different antigenic bands (16, 23, 29, 30, 40, 50, 58, 100 and 115 kD). These same antigens were recognized by serum from a patient with systemic lupus erythematosus. The binding of visceral leishmaniasis serum antibodies to ribonucleoproteins was inhibited by prior incubation of serum with either leishmanial membrane antigens, from four different species of Leishmania, or intact cells of Leishmania donovani, implying molecular resemblance between common leishmanial antigens and ribonuclear antigens. It seems that appearance of autoantibodies to ribonucleoproteins in sera of patients infected with Leishmania is not only due to simply polyclonal activation of lymphocytes, but is also the result of a molecular mimicry between leishmanial antigens and ribonucleoproteins.