G-quadruplex ligand RHPS4 radiosensitizes glioblastoma xenograft in vivo through a differential targeting of bulky differentiated- and stem-cancer cells.

BACKGROUND: Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. METHODS: RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. RESULTS: RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. CONCLUSIONS: We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

Development and validation of a near infrared spectrophotometric method to determine total antioxidant activity of milk.

In the present study a spectrophotometric method for the determination of total antioxidant activity (TAA) based on ABTS assay was developed and validated on raw milk (RM), whole UHT milk (WUM), partially skimmed UHT milk (SUM), whole pasteurised milk (WM) and partially skimmed pasteurised milk (SM). The most suitable solvent for antioxidant extraction was 80% acetone. Regardless of the type of milk, the coefficient of determination from the linearity test was greater than 0.95. The limit of detection ranged from 0.74 to 6.07µmoll-1 Trolox equivalents. Repeatability, calculated as relative standard deviation of twenty measurements within a day, and reproducibility, calculated as relative standard deviation of sixty measurements across three days, ranged from 1.24 to 4.04% and from 2.18 to 3.52%, respectively. Preservative added to RM had negligible effects on the TAA of milk. The greatest TAA was measured for SM followed by SUM, RM, WM and WUM.

PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL.

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Effects of the ruthenium-based drug NAMI-A on the roles played by TGF-ß1 in the metastatic process.

The ruthenium-based drug NAMI-A, characterised by its selectivity against solid tumour metastases, promotes TGF-ß1-dependent fibrosis and the reduction of the release of MMPs in the primary tumour. The aim of the study was to examine the interaction of NAMI-A with TGF-ß1 in the process of metastasis formation. NAMI-A (1) affects the secretion of TGF-ß1 in metastatic MDA-MB-231 cells rather than in non-tumorigenic HBL-100 cells, (2) prevails over TGF-ß1 with regard to the invasive capacity of the treated cells, and (3) contrasts integrin-dependent migration stimulated by TGF-ß1. It, thus, appears that the effects of NAMI-A on cell invasion and migration are best summarised as an interference with TGF-ß1 and a reduction of its activity in these events. At a molecular level, the similar activity of NAMI-A and TGF-ß1 on RhoA GTPase supports its interaction with cell surface integrins while TGF-ß1 can activate it by interaction with its TGFßR receptor. The inhibition of TGF-ß1-induced migration of MDA-MB-231 cells by NAMI-A cannot simply be attributed to a modulation of the Smad2 and p38MAPK pathways. In conclusion, the effects of NAMI-A on the biological role of TGF-ß1 in cancer metastasis are insufficient to attribute the responsibility for the anti-metastatic activity of the ruthenium-based drug to this target alone.

Short communication: characterization of soluble thiols in bovine milk.

Antioxidants are molecules essential for the maintenance of cell homeostasis and their intake through the diet has positive effects on human health. Among antioxidants, low-molecular-weight (LMW) thiols represent an important class of compounds. The aim of this study was to identify LMW thiols in bovine milk. A total of 96 individual milk samples from Brown Swiss, Holstein-Friesian, Alpine Grey, and Simmental cattle breeds were collected in 8 herds. The LMW thiols were extracted from the soluble fraction of milk and, following a derivatization protocol, they were separated by reverse phase HPLC and detected fluorimetrically. Six thiol species were detected and 2, glutathione (GSH) and cysteine-glycine (Cys-Gly), were identified and quantified. Regardless of the breed, the average concentration of Cys-Gly in milk was greater than that of GSH. Overall, milk from dual-purpose breeds (Simmental and Alpine Grey) was richer in LMW thiols than milk from dairy cows (Holstein-Friesian and Brown Swiss). Glutathione and Cys-Gly, closely linked metabolically, were strongly correlated. Pearson correlations of Cys-Gly with protein and casein contents were moderately low, and no relationship was found between GSH and milk chemical composition. Future research should focus on the identification of all detected LMW thiol species.

MPP(+) -dependent inhibition of Ih reduces spontaneous activity and enhances EPSP summation in nigral dopamine neurons.

BACKGROUND AND PURPOSE: 1-Methyl-4-phenylpyridinium (MPP(+) ), a potent parkinsonizing agent in primates and rodents, is a blocker of mitochondrial complex I, therefore MPP(+) -induced parkinsonism is believed to depend largely on mitochondrial impairment. However, it has recently been proposed that other mechanisms may participate in MPP(+) -induced toxicity. We tackled this issue by probing the effects of an acute application of MPP(+) on substantia nigra pars compacta (SNc) dopamine (DA) neurons. EXPERIMENTAL APPROACH: The effects of MPP(+) on SNc DA neurons in acute midbrain slices were investigated with electrophysiology techniques. KEY RESULTS: MPP(+) (50 µM) was able to (i) hyperpolarize SNc DA neurons by ∼6 mV; (ii) cause an abrupt and marked (over 50%) reduction of the spontaneous activity; and (iii) inhibit the hyperpolarization-activated inward current (Ih ). MPP(+) shifted Ih activation curve towards negative potentials by ∼11 mV both in Wistar rats and in C57/BL6 mice. Inhibition was voltage- and concentration-dependent (Imax = 47%, IC50 = 7.74 µM). MPP(+) slowed Ih activation kinetics at all potentials. These effects were not dependent on (i) block of mitochondrial complex I/fall of ATP levels; (ii) activation of type 2 DA receptor; and (iii) alteration of cAMP metabolism. Finally, MPP(+) -dependent inhibition of Ih facilitated temporal summation of evoked EPSPs in SNc DA, but not in CA1 hippocampal neurons. CONCLUSION AND IMPLICATIONS: Reduced functionality of Ih in SNc DA neurons, via increased responsiveness towards synaptic excitation, might play a role in MPP(+) -induced parkinsonism and, possibly, in the pathogenesis of human Parkinson's.

Management of corrosive injuries of the upper gastrointestinal tract. Our experience in 58 patients.

BACKGROUND: The ingestion of caustic substances is one of the most difficult conditions to be treated in Emergency Department. PATIENTS AND METHODS: The medical records of patients with caustic ingestion and hospitalized from 2003 to 2008 at the Division of General Emergency Surgery with Polyspecialistic Observation of AORN "A. Cardarelli "in Naples, have been revalued. RESULTS: From 2003 to 2008, 58 patients with caustic ingestion were admitted to our Division. Ten of these patients (17.24%) underwent surgery. Six patients underwent oesophageal and gastric resection with cervical esophagostomy and alimentary digiunostomy in emergency; two underwent exploratory laparotomy, two had gastroenteroanastomosis for antropyloric stenosis. One patient underwent new operation for a complication. In total, three reconstructions of oesophagus with colon were performed . Of the six patients undergoing esofagogastrectomy, two died in the first postoperative day, but four have passed the acute phase. CONCLUSIONS: There is no universally accepted diagnostic and therapeutic procedure for the management of these patients, who are often left - as it appears in literature - to the personal experience of the surgeon who is dealing with this situation.

Characterization of HuH6, Hep3B, HepG2 and HLE liver cancer cell lines by WNT/ß - catenin pathway, microRNA expression and protein expression profile.

Somatic mutations in the genes members of WNT/ß-catenin pathway, especially in CTNNB1 codifying for ß-catenin, have been found to play an important role in hepatocarcinogenesis. The purpose of this work is to characterize alterations of the WNT/ß-catenin signalling pathway, and to study the expression pattern of a panel of microRNAs and proteins potentially involved in the pathogenesis of liver cancer. In this respect, the molecular characterization of the most used liver cancer cell lines HuH6, Hep3B, HepG2, and HLE, could represent a useful tool to identify novel molecular markers for hepatic tumour. A significant modulation of FZD7, NLK, RHOU, SOX17, TCF7L2, TLE1, SLC9A3R1 and WNT10A transcripts was observed in all the four liver cancer cell lines. The analysis of selected microRNAs showed that miR-122a, miR-125a and miR-150 could be suitable candidates to discriminate tumoural versus normal human primary hepatocytes. Finally, Grb-2 protein expression resulted to be increased more than two-fold in liver cancer cell lines in comparison to normal human primary hepatocytes. These advances in the knowledge of molecular mechanisms involved in the pathogenesis of liver cancer may provide new potential biomarkers and molecular targets for the diagnosis and therapy.

In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model.

We have compared the organometallic arene complexes [(eta(6)-biphenyl)M(ethylenediamine)Cl](+) RM175 (M=Ru(II)) and its isostructural osmium(II) analogue AFAP51 (M=Os(II)) for their ability to induce cell detachment resistance from fibronectin, collagen IV and poly-l-lysine, and cell re-adhesion after treatment, their effects on cell migration and cell viability, on matrix metalloproteinases production, and on primary tumour growth of MCa mammary carcinoma, the effect of human serum albumin on their cytotoxicity. There are differences between ruthenium and osmium. The Os complex is up to 6x more potent than RM175 towards highly-invasive breast MDA-MB-231, human breast MCF-7 and human epithelial HBL-100 cancer cells, but whereas RM175 was active against MCa mammary carcinoma in vivo and caused metastasis reduction, AFAP51 was not. Intriguingly the presence of human serum albumin in the growth medium enhanced the cytotoxicity of both compounds. RM175 increased the resistance of MDA-MB-231 cells to detachment from substrates and both compounds inhibited the production of MMP-2. These data confirm the key role of ruthenium itself in anti-metastatic activity. It will be interesting to explore the activity of osmium arene complexes in other tumour models and the possibility of changing the non-arene ligands to tune the anticancer activity of osmium in vivo.

Inhibitory Effects of the Ruthenium Complex KP1019 in Models of Mammary Cancer Cell Migration and Invasion.

The effects of indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019, or FFC14A), the second ruthenium compound that entered clinical trials, in an in vitro model of tumour invasion and metastasis show that the antitumour effects of this compound might include also the modulation of cell behaviour although its cytotoxicity appears to be predominant over these effects. The comparison with its imidazole analogue KP418 shows however its superiority, being able to control in vitro cell growth and in some instances also in vivo tumour development. These results suggest that the activity of KP1019 is predominantly due to direct cytotoxic effects for tumour cells, evident also in vivo on primary tumour growth and that the effects on modulation of the biological behaviour of the cancer cell can be present but might have only a partial role.

Targeting ion channels in cancer: a novel frontier in antineoplastic therapy.

Targeted therapy is considerably changing the treatment and prognosis of cancer. Progressive understanding of the molecular mechanisms that regulate the establishment and progression of different tumors is leading to ever more specific and efficacious pharmacological approaches. In this picture, ion channels represent an unexpected, but very promising, player. The expression and activity of different channel types mark and regulate specific stages of cancer progression. Their contribution to the neoplastic phenotype ranges from control of cell proliferation and apoptosis, to regulation of invasiveness and metastatic spread. As is being increasingly recognized, some of these roles can be attributed to signaling mechanisms independent of ion flow. Evidence is particularly extensive for K(+) channels. Their expression is altered in many primary human cancers, especially in early stages, and they frequently exert pleiotropic effects on the neoplastic cell physiology. For instance, by regulating membrane potential they can control Ca(2+) fluxes and thus the cell cycle machinery. Their effects on mitosis can also depend on regulation of cell volume, usually in cooperation with chloride channels. However, ion channels are also implicated in late neoplastic stages, by stimulating angiogenesis, mediating the cell-matrix interaction and regulating cell motility. Not surprisingly, the mechanisms of these effects are manifold. For example, intracellular signaling cascades can be triggered when ion channels form protein complexes with other membrane proteins such as integrins or growth factor receptors. Altered channel expression can be exploited for diagnostic purposes or for addressing traceable or cytotoxic compounds to specific neoplastic tissue. What is more, recent evidence indicates that blocking channel activity impairs the growth of some tumors, both in vitro and in vivo. This opens a new field for medicinal chemistry studies, which can avail of the many available tools, such as blocking antibodies, antisense oligonucleotides, small interfering RNAs, peptide toxins and a large variety of small organic compounds. The major drawback of this approach is that some ion channel blockers produce serious side effects, such as cardiac arrhythmias. Therefore, drug developing efforts aimed at producing less harmful compounds are needed and we discuss possible approaches toward this goal. Finally, we propose that a novel therapeutic tactic could be developed by unlocking ion channels from multiprotein membrane signaling complexes.

Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast.

The molecular properties and subcellular location of bound gamma-glutamyl transferase (GGT) were studied, and an experimental setup devised to assess its functions in barley roots. Enzyme histochemistry was used to detect GGT activity at tissue level; immunocytochemistry to localize the protein at subcellular level; and modelling studies to investigate its surface charge properties. GGT activity in vivo was measured for the first time. Functions were explored by applying chemical treatments with inhibitors and the thiol-oxidizing drug diamide, performing time-course chromatographic and spectrophotometric analyses on low-molecular-weight thiols. Gamma-glutamyl transferase activity was found to be high in the root apical region and the protein was anchored to root cell wall components, probably by basic amino acid residues. The results show that GGT is essential to the recovery of apoplastic glutathione provided exogenously or extruded by oxidative treatment. It is demonstrated that GGT activity helps to salvage extracellular glutathione and may contribute to redox control of the extracellular environment, thus providing evidence of a functional role for gamma-glutamyl cycle in roots.

A case report of a patient with microcephaly, facial dysmorphism, chromosomal radiosensitivity and telomere length alterations closely resembling "Nijmegen breakage syndrome" phenotype.

Genetic heterogeneity in Nijmegen breakage syndrome (NBS) is highlighted by patients showing clinical and cellular features of NBS but with no mutations in NBS1 and normal levels of nibrin. NBS is an autosomal recessive disorder, whose clinical cellular signs include growth and developmental defects, dysmorphic facies, immunodeficiency, cancer predisposition, chromosomal instability and radiosensitivity. NBS is caused by mutations in the NBS1 gene, whose product is part of the MRE11/RAD50/NBS1 complex involved in the DNA double-strand break (DSB) response pathway. Since the identification of the NBS1 gene, patients with NBS clinical signs, particularly severe congenital microcephaly, are screened for mutations in the NBS1 gene. Further analyses include X-ray-induced chromosome aberrations, telomere analysis, kinetics of DSBs repair, levels of a panel of proteins involved in the maintenance of genetic stability, radiation-induced phosphorylation of various substrates and cell cycle analysis. We describe a patient with a NBS clinical phenotype, chromosomal sensitivity to X-rays but without mutations in the whole NBS1 or in the Cernunnos gene. Enhanced response to irradiation was mediated neither by DSBs rejoining defects nor by the NBS/AT-dependent DNA-damage response pathway. Notably, we found that primary fibroblasts from this patient displayed telomere length alterations. Cross-talk between pathways controlling response to DSBs and those involved in maintaining telomeres has been shown in the present patient. Dissecting the cellular phenotype of radiosensitive NBS-like patients represents a useful tool for the research of new genes involved in the cellular response to DSBs.

Synthesis, conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe.

For-Met-betaAlapsi[CSNH]-Phe-OMe (3), For-Met-betaAlapsi[CH2NH]-Phe-OMe (5), For-Met-NH-pC6H4-SO(2-Phe-OMe 8a), For-Met-NH-mCH4-SO2-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. (1)H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated beta-residue.

Integrated neuroendocrine immune risk factors in relation to rheumatoid arthritis: should rheumatologists now adopt a model of a multiyear, presymptomatic phase?

An integrative perspective of neuroendocrine immune (NEI) and related risk factors for the onset of rheumatoid arthritis (RA) is presented, based upon studies of the long-term presymptomatic phase. Besides the recognized genetic markers and familial predisposition, multiple immunological precursors of RA have been identified many years before the clinical onset of inflammatory manifestations. Rheumatoid factors and related antibodies occur in approximately one-half of presymptomatic susceptibles. Cigarette smoking in sufficient amount and duration is a major risk factor for RA, particularly for postmenopausal-onset women and for men. In premenopausal-onset RA, subtle insufficiency of adrenal cortical function is less well recognized. In such women, cytokine imbalance may also precede inflammatory onset of RA. In males alone, multiple hormonal and cytokine correlations were found many years before the onset of RA, implying long-term activation or perturbation of this NEI system. The proposed physiopathogenetic model of RA requires further controlled, prospective studies for validation of the multiyear presymptomatic phase of RA. Such studies promise to clarify the currently unknown causal and sequential chains in this enigmatic disease.

hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines.

Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K(+) channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K(+) channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1's biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K(+) channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies.

[Laparoscopic incisional hernia repair as first therapeutic choice]. / L'approccio laparoscopico nella riparazione del laparocele quale prima scelta terapeutica.

AIM: Incisional hernias are one of the most frequent complications of open abdominal surgery. Historically, the best results have been obtained with the open rives-stoppa approach. This is done by fixing a large piece of prosthetic mesh behind the rectus muscle. Laparoscopic approach allows similar mesh placement with minimal dissection and lower recurrence rate compared to the open mesh repair. METHODS: Between October 2001 to September 2003, 75 consecutive patients were scheduled to undergo laparoscopic incisional hernia repair with ePTFE mesh (Gore-Tex Dualmesh Plus). Postoperative complications were recorded and analysed. RESULTS: Most were obese affected by multiple wall defects Conversion to open surgery was required in 1 case Postoperative complications occurred 13.3%. Recurrence occurred in one only case. CONCLUSIONS: The key to the success of this procedure is avoidance of complications. The laparoscopic approach is safe, effective and relatively complication-free option in the management of patients presenting with a first time or recurrent incisional hernia and recommended as the treatment of choice.

Screening of lung cancer with low dose spiral CT: results of a three year pilot study and design of the randomised controlled trial ''Italung-CT''.

PURPOSE: To report the results of a three-year observational pilot study of lung cancer screening with low dose computed tomography (CT) and to present the study design of a randomised clinical trial named as ''Italung-CT''. MATERIALS AND METHODS: Sixty (47 males and 13 females, mean age 64+/-4.5 years) heavy smokers (at least 20 packs-year) underwent three low-dose spiral CT screening tests one year apart on a single slice or multislice CT scanner. Indeterminate nodules were managed according to the recommendations of the Early Lung Cancer Action Project. RESULTS: and Indeterminate nodules were observed in 33 (55%) of the subjects (60% at the baseline screening test, 24% at the first annual test and 16% at the second annual test). The size of the largest indeterminate nodule was <5 mm in diameter in 20 subjects, 10 of whom showed the nodule at the baseline test. Forty-five subjects (75%) completed the first annual test and 42 (70%) the second annual test. One (1.6%) prevalent lung cancer (adenosquamous carcinoma) and one (2.2%) incident lung cancer (small cell cancer at the first annual examination) were observed, as well as a pulmonary localisation of Hodgkin's lymphoma (at the second annual test). In addition, one subject underwent lung surgery for a chondromatous hamartoma. CONCLUSIONS: The results of the pilot study are substantially in line with those of other observational studies of greater sample size. This justifies optimism about the reliability of the results in the screened arm of the ''Italung CT'' trial which has just begun.

Genetic heterogeneity for a Nijmegen breakage-like syndrome.

Nijmegen breakage syndrome (NBS) is a rare, autosomal-recessive chromosome instability disorder characterized by growth and developmental defects, immunodeficiency, high susceptibility to lymphoid malignancies, hypersensitivity to ionizing radiation and aberrant cell-cycle checkpoint control. The disease is caused by mutations in the NBS1 gene, which encodes nibrin, a component of the hMre11-Rad50-p95 complex involved in cellular response to DNA double-strand breaks. Genetic heterogeneity has been suggested in at least two patients with the NBS phenotype, but no mutation in the NBS1 gene; recently, mutations in the gene encoding the enzyme ligase IV have been identified in patients with signs of NBS. We describe a boy with an NBS clinical phenotype but no mutation in either the NBS1 or the LIG4 genes. The analysis of his cellular phenotype reveals chromosome instability and radiosensitivity, but normal cell-cycle checkpoint control. In addition, a literature review was carried out to summarize and compare data of all NBS-like patients reported to date. This case confirms genetic heterogeneity for NBS. We believe that dissecting the clinical and cellular phenotypes of this and other NBS-like patients will provide useful information for the research of new genes involved in cellular response to DNA damage and the assessment of cancer risk in NBS-like syndrome.