[Cancer screening programme in health care workers]. / Applicazione di screening antitumorali in una popolazione di operatori sanitari.

From the 2002 through 2009 years 419 health care workers of the Hospital of Lecco, occupationally exposed to X-ray, were invited to undergo a cancer screening programme for the early diagnosis of cervical, breast, colorectal and prostate cancers. A total of 341 subjects performed the screening tests with an overall compliance of 83,8%; the participation rate to each test was significantly higher than that of general population. Breast cancer was diagnosed and treated in 5 women, cervical premalignant lesions in 8 women and colorectal adenomas in 13 subjects; no prostate cancer was detected. The participation rate, the premalignant and malignant findings and the cost-effectiveness analysis are consistent with the possibility that cancer screening programme can be set out as health promotion activity in health care workers.

Antigen processing of vesicular stomatitis virus in situ. Interdigitating dendritic cells present viral antigens independent of marginal dendritic cells but fail to prime CD4(+) and CD8(+) T cells.

Acute macrophage (M phi) depletion, using a liposome-mediated 'suicide technique', markedly suppressed priming of splenic CD4(+) and CD8(+) T-cell responses to vesicular stomatitis virus (VSV). However, phagocytic marginal dendritic cells (MDC), but not interdigitating dendritic cells (IDC), are now known to be also depleted by this technique. To clarify the role splenic dendritic cell (DC) subsets and M phi play in priming for a virus-specific T-cell-mediated immune response, DC and M phi were purified from VSV-infected mice and assayed for the presence of epitopes recognized by VSV helper T (Th) cells and cytotoxic T lymphocytes (CTL). Antigen pulse experiments performed in situ demonstrated that VSV Th cell and CTL epitopes became transiently associated only with DC, but not M phi or B cells, indicating that DC represent the critical antigen-presenting cell (APC) population in vivo for this virus. The failure of MDC/M phi-deficient mice to become primed was not due to the complete elimination of antigen-presenting DC because VSV peptide/class I and II complexes were detected on IDC following lipsome-mediated elimination of phagocytic cells. However, the VSV-induced chemokine response was dramatically suppressed in these mice. Thus, despite the expression of VSV peptide/class I and II complexes, IDC are not sufficient to prime VSV Th cells in the absence of MDC and/or splenic M phi.

Three subpopulations of fast axonally transported retinal ganglion cell proteins are differentially trafficked in the rat optic pathway.

Post-Golgi trafficking of the major fast axonally transported (FT) proteins was investigated in the rat optic pathway. Following intra-ocular injection of 35S-methionine, radiolabeled FT proteins in the optic tract (OT) and superior colliculus (SC) were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Twenty FT proteins, including a known plasma membrane protein (SNAP-25) and synaptic vesicle protein (synaptobrevin-2), displayed consistent 2D-PAGE migration behavior and were chosen for densitometric quantitative analysis. Results showed that at least three subpopulations of the 20 FT proteins could be differentiated based on their trafficking behavior to axons (OT) vs. terminals (SC). To assess whether Golgi-independent processes (e.g., delayed somal release and/or retrograde transport) could account for the differential compartmentation behavior between the three FT classes, we assessed whether radiolabeled FT proteins became redistributed in the optic pathway following a nerve transection blockade. The results showed that radiolabelled FT proteins did not show a quantitative change in their axon vs. terminal compartmentation in response to disconnection from cell bodies or targets. Thus, the three classes of fast axonally transported proteins were likely trafficked to distinct destinations in the optic pathway by Golgi sorting mechanisms.

Bleomycin-induced chromosome aberrations in lymphocytes derived from patients with lamellar ichthyosis.

Patients affected by some genetic skin defects, for example, dyskeratosis congenita or scleroderma, may present spontaneous or induced chromosomal fragility. Hence we performed a cytogenetic analysis in families of patients affected by lamellar ichthyosis, an autosomal recessive disease not yet fully characterized at the cellular and molecular levels. Chromosomal fragility was assayed in untreated lymphocyte cultures and in those supplemented with aphidicolin or bleomycin. Cells from some affected patients and some of their parents showed hypersensitivity to the radiomimetic agent bleomycin.

A new case of Ambras syndrome associated with a paracentric inversion (8) (q12; q22)

Ambras syndrome (AS) is a special form of congenital universal hypertrichosis described for the first time by Baumeister et al. (1). This form differs from other forms of congenital hypertrichosis in the pattern of hair distribution and its associated anomalies. The molecular-genetic cause of AS is unknown; the association of AS with a pericentric inversion (8) (p11.2; q22) described in the case of Baumeister so far has been unique in the literature. This report is the tenth with clinical signs of AS so far described in the literature and the second with an inversion in chromosome 8 and the first with evaluation of peripheral androgens. The new-born girl presented with abundant and dark hair on the face and ears, on the shoulders and on the arms; the other parts of the body were covered with fine, lightly pigmented hair. The face showed many dysmorphic features. Chromosome analysis showed a paracentric inversion of one chromosome 8. The breakpoints were localised at q12 and q22. The parental karyotypes were normal. Laboratory investigation showed normal plasma levels of testosterone, androstenedione (A), 17-hydroxyprogesterone, dehydroepiandrosterone-sulphate (DHA-S), free testosterone (FT), dihydrotestosterone (DHT) and 3alpha-androstanediol-glucuronide (3AG). Here we report a chromosomal inversion similar to that found previously not associated with alterations in androgen plasma levels.

Induction of apoptosis by bleomycin in resting and cycling human lymphocytes.

Bleomycin induces DNA and chromosome breakage. The differential sensitivity to the drug has been used in vitro to identify individuals at high risk of developing tumours. However, there are limited reports on the ability of bleomycin to induce apoptosis. In this study we tested induction of apoptosis in human peripheral lymphocytes by bleomycin at different concentrations and different culture times using various parameters, such as nuclear fragmentation and DNA fragmentation, evaluated either in situ with terminal transferase and labelled nucleotides (TUNEL) or by flow cytometry analysis. We demonstrate that bleomycin induces apoptosis without previous permeabilization of the cell membrane. Cell death occurs mainly by apoptosis and not by necrosis, with significant alteration of membrane lipoperoxidation (evaluated by luminescence).

In vitro effects of growth hormone (GH) and insulin-like growth factor I and II (IGF-I and -II) on chromosome fragility and p53 protein expression in human lymphocytes.

BACKGROUND: We have reported previously that growth hormone (GH) therapy increases cell radiosensitivity; in this study we tested whether GH itself or IGFs induce chromosome aberrations and investigated the expression of p53 protein in response to DNA damage. METHODS: Human peripheral blood lymphocytes were incubated with GH [100 and 1000 microg L(-1)], insulin-like growth factor I [IGF-I; 150 and 1000 microg L(-1)] and IGF-II [600 and 1200 microg L(-1)] for 24 h. The radiomimetic agent bleomycin [BLM; 5 microgm L(-1)] was added in the last 3 h. Cytogenetic analysis was performed by assessing the percentages of damaged cells (%DC) and chromosome aberrations (%CA). The expression of p53 was investigated by flow cytometric assay using the monoclonal antibody DO-7, and expressed as percentage positive cells and mean fluorescence intensity. RESULTS: BLM significantly increased both percentage DC and percentage CA and p53 expression (P < 0.01). The %DC was unaffected by the tested peptides. IGF-I [150 microg L(-1)] increased spontaneous percentage CA (P < 0.01). All peptides further increased the BLM-induced chromosome breakage: GH 100 and 1000 microg L(-1) by 30% and 73% respectively, IGF-I 150 and 1000 microg L(-1) by 41% and 96% respectively and IGF-II 600 and 1200 microg L(-1) by 89% and 45% respectively. The spontaneous and BLM-induced expression of p53 was unaffected by GH, whereas it was significantly increased by IGFs (P < 0001). CONCLUSIONS: These results indicate that the DNA-damaging effect of BLM is amplified by GH and, more markedly, IGF-I and -II. IGF-I and -II also stimulate p53 protein expression that, taking part in DNA repair, may counteract the IGF action on genome stability.

Sister chromatid exchanges and DNA topoisomerase II inhibitors: effect of low concentrations of etoposide (VP-16) in ataxia telangiectasia lymphoblastoid cell lines.

The correlation between etoposide (VP-16) cytotoxicity and the induction of sister chromatid exchanges (SCEs) suggested that the promotion of DNA recombination events may be crucial for the activity of antitopoisomerase drugs. To further evaluate this hypothesis, we investigated the correlation between VP-16 induction of SCEs, chromosomal aberrations and cell cycle alterations in lymphoblastoid cell lines derived from patients affected by ataxia telangiectasia (AT), whose cells are known as hypersensitive to the cytotoxic and clastogenic activity of DNA topoisomerase II inhibitors. Our present study has shown that AT homozygous and heterozygous cell lines exposed to low VP-16 concentrations, although hypersensitive to the induction of chromosomal aberrations, exhibit an induction of SCEs comparable to that found in normal cell lines. Moreover, while the clastogenic effect of the drug was directly correlated to the reduction of the mitotic index, the enhancement of SCE frequencies, obtained over the same range of VP-16 concentrations, was not paralleled by a modification of proliferation index. Thus, these results suggest that etoposide retains in AT cells a strong clastogenic and cytostatic activity which is independent from DNA recombination events and which may be important for the induction of cell death by this kind of drug.

Differential effects of axotomy on the in vivo synthesis of the stress-inducible and constitutive 70-kDa heat-shock proteins in rat dorsal root ganglia.

The purpose of this study was to the test the hypothesis that heat-shock protein expression is upregulated (or induced) in dorsal root ganglia (DRG) following axotomy. To test this hypothesis, DRG or sciatic nerve (SN) proteins were pulse-labelled in vivo with [35S]methionine and the metabolic synthesis of two major 70-kDa heat-shock proteins, the constitutive species (hsc70) and stress-inducible species (hsp68), were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Results showed that DRG hsp68 expression was absent (or barely detectable) under normal (sham-axotomy) conditions. However, following long-range axotomy (35 mm from DRG), there was a delayed (> 12 h post-axotomy) and transient upregulation of DRG hsp68 metabolic synthesis. Control studies demonstrated that, although DRG hsp68 was upregulated, hsp68 was not induced in SN regions proximal to the crush site. In contrast to DRG hsp68 expression, there was abundant DRG hsc70 synthesis under normal conditions that did not significantly change following axotomy. These results suggest that a specific stress protein response is induced in DRG following axotomy.

T cell priming against vesicular stomatitis virus analyzed in situ: red pulp macrophages, but neither marginal metallophilic nor marginal zone macrophages, are required for priming CD4+ and CD8+ T cells.

Since extensive degradation may be required to present complex Ags, we addressed whether macrophages (M phi) might function as APC for anti-viral cell-mediated immune responses. To study this question, murine splenic M phi were depleted by i.p. administration of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP-liposomes or clodronate-liposomes) before priming mice with vesicular stomatitis virus (VSV). Cl2MDP-liposome treatment resulted in the rapid (1-day) depletion of splenic M phi that was associated with a suppression of the ability of M phi-deficient mice to generate secondary anti-VSV CTL and Th cell proliferative responses in vitro. Control studies demonstrated that splenic dendritic cells were not adversely affected by treatment with Cl2MDP-liposomes. To assess the contribution of splenic M phi subpopulations to T cell priming against this virus, priming was delayed following treatment with Cl2MDP-liposomes until specific M phi subsets had repopulated the spleen. This analysis revealed that repopulation by red pulp M phi, but not with other splenic M phi subsets, was associated with the ability to mount normal secondary CTL and Th cell responses against VSV. Depletion of splenic, but not resident, peritoneal M phi by i.v. injection of Cl2MDP-liposomes did not rescue T cell priming in VSV-infected mice. Thus, only red pulp M phi, and not other splenic or peritoneal M phi populations, are necessary for T cell priming to VSV, a biochemically complex Ag.

Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident.

In a previous paper we reported that a group of children exposed to ionizing radiation following the Chernobyl accident exhibited an appreciable number of chromosome breaks and rearrangements reflecting the persistence of a radiation-induced damage. The results suggested that the children were still exposed to radioactive contamination through consumer foodstuff and life styles. In the present paper, 31 exposed children have been considered together with a control group of 11 children with the aim to confirm previous results. All children underwent whole-body counter (WBC) measures and conventional cytogenetic analysis. The frequency of chromosome aberrations detected by conventional cytogenetics in the group of children chronically exposed to low doses of ionizing radiation resulted in significant differences with respect to the control group. The present work suggests that, for these groups of children, even if the frequency of aberrations is very low and the observation of statistically significant differences is consequently a problem, a persistently abnormal cytogenetic picture is still present several years after the accident.

Do human lymphocytes exposed to the fallout of the Chernobyl accident exhibit an adaptive response? III. Challenge with bleomycin in lymphocytes from children hit by the initial acute dose of ionizing radiation.

In the present paper, we report data on the possible adaptive response, induced in vivo by exposure to ionizing radiation to a challenge treatment with the radiomimetic glycopeptide bleomycin (BLM). Lymphocytes from children living in Pripjat at the time of the Chernobyl accident, and thus hit by the initial acute dose of ionizing radiation, were treated for the last 5 h of culture with 0.004 U/ml BLM. Significantly lower chromosome damage was found only in lymphocytes from children who, independently of the initial acute exposure to ionizing radiation, still showed a 137Cs internal contamination, due to persistent continuous exposure to low doses of radiation. The present results indicate that past exposure to acute high dose of ionizing radiation does not interfere with resistance to BLM which is related to internal contamination.

Do human lymphocytes exposed to the fallout of the Chernobyl accident exhibit an adaptive response? 1. Challenge with ionizing radiation.

Several studies suggest that cells appear to become less susceptible to the induction of radiation damage, and in particular of chromosome and chromatid aberrations in short-term cultures of human lymphocytes, when a challenge exposure to ionizing radiation is preceded by a low 'adaptive' dose. Contradictory results have been reported on the conditions under which the phenomenon can be evidenced. In the present work, circulating lymphocytes of 13 children contaminated from the fallout after the Chernobyl accident were tested for their capability to exhibit an adaptive response in experiments in which the challenge dose was administered to stimulated lymphocytes in the S-G2 phase. Furthermore, the possible influence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, was also investigated. Our results indicate that, at least in the instance of the end-point here used (chromosome and chromatid aberrations, the former resulting possibly from the Cs burden), human lymphocytes, chronically exposed to low doses from fallout, do not exhibit any decreased susceptibility to ionizing radiation. However, as reported in the accompanying paper, the same samples appear to show an 'adaptive' response when exposed to a challenge treatment with bleomycin (B. Tedeschi et al., 1995, this issue).

Do human lymphocytes exposed to the fallout of the Chernobyl accident exhibit an adaptive response? 2. Challenge with bleomycin.

The present study concerns the possible adaptive response, induced in vivo by a continuous exposure to ionizing radiations, to a challenge treatment with the radiomimetic glycopeptide bleomycin (BLM). Lymphocytes from children contaminated as a consequence of Chernobyl accident were treated for the last 5 h of culture with 2.5 micrograms/ml BLM. The induced chromosome damage was significantly lower than that found with the same treatment in lymphocytes from control children. This hyposensitivity to BLM was still present if, 1 h after the addition of the drug, inhibitors of the enzymes involved in DNA repair, such as 3-aminobenzamide (2 mM), or aphidicolin (0.4 microM) or 3-dideoxythymidine (5 mM) were added to the cultures. The resistance to BLM in lymphocytes from contaminated children seems to be related to a mechanism upstream in respect to the activities of enzymes involved in the DNA repair and specifically linked to the action of this drug. This is consistent with the different response found when the cells were challenged with ionizing radiation in vitro, as reported in the accompanying paper (L. Padovani, L. et al. (1995) Mutation Res., this issue).

Characteristic chromosomal fragility of human embryonic cells exposed in vitro to aphidicolin.

The frequency and distribution of aphidicolin (APC)-induced common fragile sites (cfs) were analyzed in human embryonic cells of different origins. Embryonic lung fibroblasts (MRC-5), amniocytes (AMINO) and embryonic retina cells (HERO790) are as sensitive to the APC-induced clastogenic effect as peripheral lymphocytes, whereas embryonic kidney cells (HEK) seem more resistant to the induction of chromosomal gaps and breaks by the drug. Analysis of the distribution of fragile sites confirmed that the expression of specific APC-induced cfs varies in different cells and that the embryonic cell strains show a greater similarity among themselves than to lymphocytes. In addition, HEK, MRC-5, HERO790 and AMINO cells show specific APC induction of the cfs at the 1p31.2 chromosomal band, which seems to be a distinctive feature of the embryonic stage of cells.

Complex glycerol kinase deficiency: an unusual cause of salt-wasting in males.

We report the case of a male infant who at 10 days of life presented with salt-wasting. Congenital adrenal hyperplasia was excluded on the basis of normal 17 alpha-hydroxy-progesterone plasma levels evaluated before the onset of steroid replacement therapy. The incidental finding of hypertriglyceridaemia led us to suspect the condition of complex glycerol kinase deficiency which was confirmed by the direct measurement of serum glycerol (7.16 mmol/l, normal range 0.02-0.21). Serum creatine kinase was markedly elevated (5963 U/l, normal range 37-290). High resolution cytogenetic investigation of peripheral blood showed a small interstitial deletion within Xp21. The same deletion was found in the patient's mother although not in his maternal grandmother. We present this case in order to emphasize the necessity of evaluating plasma triglycerides in all neonatal males with salt-wasting which can not be explained by congenital adrenal hyperplasia. Plasma triglycerides measurement carried out using a routine clinical method which measures glycerol released after lipolysis facilitates early recognition of this syndrome, and enables appropriate therapy and subsequent genetic counselling.

Subregional localization of 14 yeast artificial chromosomes to human chromosome region 1p by fluorescence in situ hybridization.

We have sublocalized to the region between 1p22 and 1p33 a total of 14 yeast artificial chromosomes previously assigned to a broader area of human chromosome 1p. Our purpose was to map DNA sequences that could be used for the molecular characterization of the two common fragile sites present in bands 1p31.2 and 1p32, the expression of which is increased in patients with neuroblastomas.

Priming antiviral cytotoxic T lymphocytes: requirement for CD4+ cells is dependent on the antigen presenting cell in vivo.

We have analyzed cytotoxic thymus-derived lymphocyte (CTL) responses to vesicular stomatitis virus (VSV) to determine whether VSV precursor CTL (pCTL) can be primed in vivo in the absence of CD4+ cells. Our studies demonstrated that secondary anti-VSV CTL responses in vitro were markedly reduced by CD4-depletion prior to priming in vivo with VSV. Limiting dilution analysis indicated that the vast majority (> 90%) of VSV pCTL failed to become primed when exposed to VSV in the absence of CD4+ cells. A second minor population (5-10%) of pCTL was identified that was reproducibly primed in CD4-deficient mice. In contrast to CD4-depleted mice infected with free, infectious virus, CD4-deficient mice primed with VSV-infected, activated B cells mounted normal secondary anti-VSV CTL responses in vitro. Precursor estimates indicated that virtually all VSV pCTL became primed using this cellular immunogen. CD4-independent priming could not be achieved using VSV-infected, activated T cells, another permissive cell type for VSV replication. Thus, most VSV pCTL require inductive signals from classical CD4+ helper T cells in order to become primed in vivo and this requirement may be regulated in vivo by the antigen presenting cell.

Heat stress induces hsc70/nuclear topoisomerase I complex formation in vivo: evidence for hsc70-mediated, ATP-independent reactivation in vitro.

We previously demonstrated that in murine T cells thermotolerance correlated with heat shock protein 70 (hsp70) synthesis and protection of nuclear type I topoisomerase (topo I). Topo I activity returned to normal levels following heat stress even in cells not rendered thermotolerant by a prior heat shock. Recovery of topo I activity was not dependent on de novo protein synthesis, suggesting that the cell possesses a pathway(s) for refolding this nuclear protein. In this report we demonstrate that topo I and hsc70, the constitutively produced member of the hsp70 family, associated in vivo during heat stress. That this association may play a physiologically important role in protecting topo I activity from heat stress was suggested by the observation that hsc70 protected topo I from heat inactivation in vitro. hsc70 but not actin also reactivated previously heat-denatured topo I in a dose-dependent fashion. However, refolding of heat-denatured topo I by purified hsc70 was inefficient relative to a hsc70-containing cell lysate. Protection from heat inactivation as well as reactivation by hsc70 did not require exogenous ATP. Similarly, reactivation by the cell lysate was not inhibited by ADP or a nonhydrolyzable analogue of ATP. Thus, our studies suggest that nuclear topo I complexes with hsc70 during heat stress, which may explain, at least in part, why hsp70 proteins accumulate in the nucleus, particularly the nucleolus. This interaction may limit heat-induced protein damage and/or accelerate restoration of protein function in an ATP-independent reaction.

Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident.

The present study concerns the monitoring of children from the Byelorussian, Ukrainian and Russian republics exposed to the fall-out of the Chernobyl accident. Cytogenetic analyses have been performed on 41 children coming from different areas and exhibiting varying amounts of 137Cs internal contamination, as evaluated by whole-body counter (WBC) analysis. On a total of 28,670 metaphases scored, radiation-induced chromosome damage is still present, although at a very low frequency. Due to the very low fraction of dicentrics, because of the time elapsed from the accident and the relatively low doses of exposure, radiobiological dosimetry is not possible for these children. However, considering that the WBC data indicate that the children are still exposed to 137Cs contamination, the observed occurrence of stable chromosome rearrangements and breaks may represent the persisting effect of continuous low doses of radiation. The present study also indicates that the parallel use of internal contamination dosimetry and cytogenetics could be usefully employed to monitor individual exposure to radiation and to define further management measures.