Showing papers by "Casa Sollievo della Sofferenza published in 2001"

Reelin gene alleles and haplotypes as a factor predisposing to autistic disorder

Abstract: Autistic disorder (MIM 209850) is currently viewed as a neurodevelopmental disease. Reelin plays a pivotal role in the development of laminar structures including the cerebral cortex, hippocampus, cerebellum and of several brainstem nuclei. Neuroanatomical evidence is consistent with Reelin involvement in autistic disorder. In this study, we describe several polymorphisms identified using RNA-SSCP and DNA sequencing. Association and linkage were assessed comparing 95 Italian patients to 186 ethnically-matched controls, and using the transmission/disequilibrium test and haplotype-based haplotype relative risk in 172 complete trios from 165 families collected in Italy and in the USA. Both case-control and family-based analyses yield a significant association between autistic disorder and a polymorphic GGC repeat located immediately 5' of the reelin gene (RELN) ATG initiator codon, as well as with specific haplotypes formed by this polymorphism with two single-base substitutions located in a splice junction in exon 6 and within exon 50. Triplet repeats located in 5' untranslated regions (5'UTRs) are indicative of strong transcriptional regulation. Our findings suggest that longer triplet repeats in the 5'UTR of the RELN gene confer vulnerability to autistic disorder.

Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase -- increased expression with reduced muscle damage.

Abstract: The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.

Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates.

Abstract: To determine the frequency and distribution of mitochondrial DNA mutations in breast cancer, 18 primary breast tumors were analyzed by direct sequencing. Twelve somatic mutations not present in matched lymphocytes and normal breast tissues were detected in 11 of the tumors screened (61%). Of these mutations, five (42%) were deletions or insertions in a homopolymeric C-stretch between nucleotides 303-315 (D310) within the D-loop. The remaining seven mutations (58%) were single-base substitutions in the coding (ND1, ND4, ND5, and cytochrome b genes) or noncoding regions (D-loop) of the mitochondrial genome. In three cases (25%), the mutations detected in coding regions led to amino acid substitutions in the protein sequence. We then screened an additional 46 primary breast tumors with a rapid PCR-based assay to identify poly-C alterations in D310, and we found seven more cancers with alterations. Using D310 mutations as clonal marker, we detected identical changes in five of five matched fine-needle aspirates and in four of four metastases-positive lymph nodes. The high frequency of D310 alterations in primary breast cancer combined with the high sensitivity of the PCR-based assays provides a new molecular tool for cancer detection.

Identification of a mononucleotide repeat as a major target for mitochondrial DNA alterations in human tumors

Abstract: Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.

Longitudinal evaluation of vitamin D status in healthy subjects from southern Italy: seasonal and gender differences.

Abstract: Vitamin D status is currently considered among the relevant determinants of skeletal integrity. Since vitamin D levels present seasonal variations, we longitudinally studied young healthy men and women in order to investigate the related physiologic modifications of both calcium homeostasis and bone remodeling. Thirty-two men (mean age 39.4 ± 7.8 years) and 58 premenopausal women (aged 36.9 ± 6.4 years) from southern Italy were studied. In all subjects the following parameters were measured both in winter and in summer: serum calcium, phosphorus, creatinine, total alkaline phosphatase activity, 25-hydroxyvitamin D (25OHD), parathyroid hormone (PTH), osteocalcin (BGP), together with urinary calcium (Ca/Cr), total pyridinoline (Pyr/Cr) and deoxypyridinoline (d-Pyr/Cr), corrected for creatinine excretion. In both sexes 25OHD levels were significantly higher in summer, while PTH values were lower, than in winter. The prevalence of hypovitaminosis D, defined by concentrations of 25OHD lower than 30 nmol/l, was 17.8% in winter and 2.2% in summer in the whole sample, while it was 27.8% and 3.4%, respectively, among female subjects. Indeed male subjects did not display hypovitaminosis D, having throughout the year significantly higher calcium and 25OHD levels together with lower PTH values, than the women. Moreover, alkaline phosphatase total activity was more elevated in men both in winter and in summer. In women, during winter, bone remodeling markers levels were higher while urinary calcium levels were lower than in summer. In the whole sample serum 25OHD correlated positively with serum calcium and inversely with PTH. The seasonal percentage variations in PTH were inversely correlated with those of Ca/Cr. Our results show a relatively high prevalence of subclinical vitamin D deficiency among young healthy women from southern Italy. Significant gender-specific differences have been demonstrated in both calcium homeostasis and skeletal remodeling indexes; the seasonal fluctuations in the vitamin D–PTH axis are accompanied by cyclical variations of bone turnover rate, which were more pronounced in women.

The Q allele variant (GLN121) of membrane glycoprotein PC-1 interacts with the insulin receptor and inhibits insulin signaling more effectively than the common K allele variant (LYS121).

Abstract: When overexpressed, the membrane glycoprotein PC-1 may play a role in human insulin resistance through the inhibition of insulin receptor (IR) autophosphorylation. A PC-1 variant (K121Q, with lysine 121 replaced by glutamine) is also associated with whole-body insulin resistance when not overexpressed. To better understand the effects of the Q allele on IR function and downstream signaling, we transfected cultured cells with cDNAs for either the Q or the K alleles. In human MCF-7 cells, the Q allele was severalfold more effective (P < 0.05-0.01) than the K allele in reducing insulin stimulation of IR autophosphorylation, insulin receptor substrate-1 phosphorylation, phosphatidylinositol 3-kinase activity, glycogen synthesis, and cell proliferation. Similar data on IR autophosphorylation inhibition were also obtained in mouse R-/hIR and human HEK 293 cell lines. In transfected MCF-7 cells, 125I-labeled insulin binding and IR content were unchanged, and PC-1 overexpression did not influence IGF-1 stimulation of IGF-1 receptor autophosphorylation. Both the Q and K alleles directly interacted with the IR, as documented by coimmunoprecipitation assays. This interaction was greater for the Q allele than for the K allele (P < 0.01), suggesting that direct PC-1-IR interactions are important for the PC-1 inhibitory effect on insulin signaling. In conclusion, the Q allele has stronger inhibitory activity on IR function and insulin action than the more common K allele, and this is likely a consequence of the intrinsic characteristics of the molecule, which more strongly interacts with the IR.

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.

Abstract: Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Risk of Pregnancy-related Venous Thrombosis in Carriers of Severe Inherited Thrombophilia*

Abstract: Homozygous carriers of factor V Leiden have an approximately 80-fold increased risk of venous thrombosis. Also double heterozygous carriers of both the factor V Leiden and the prothrombin gene mutations are at high thrombotic risk. The magnitude of the risk of venous thrombosis in pregnant women with the two severe thrombophilic conditions has not been estimated so far. We performed a multicenter retrospective family study in women with homozygous factor V Leiden, double heterozygous factor V Leiden and the prothrombin gene mutation, and women with normal coagulation. Only relatives of index patients with thrombosis formed the study cohort. Fifteen homozygous and 39 double heterozygous women were compared to 182 women with normal coagulation. Venous thrombosis occurred in 3 of 19, 2 of 50 and 1 of 221 pregnancies, respectively. One thrombotic episode occurred in the third trimester, the remaining 5 in the postpartum. The prevalence of venous thrombosis was 15.8% (95% CI 3.4-39.6) for homozygotes, 4.0% (95% CI 0.5-13.7) for double heterozygotes and 0.5% for women with normal coagulation. The relative risk of pregnancy-related venous thrombosis was 41.3 (95% CI 4.1-419.7) for homozygous and 9.2 (95% CI 0.8-103.2) for double heterozygous carriers. In conclusion, homozygous carriers of factor V Leiden and, to a lesser extent, double heterozygous carriers of factor V Leiden and of the prothrombin mutation have an increased risk of venous thrombosis during pregnancy, particularly high during the postpartum period. On the basis of these findings we recommend that these women receive anticoagulant prophylaxis at least in the postpartum, that should perhaps be extended to the whole pregnancy in homozygous carriers.

Familial thrombophilia and the occurrence of fetal growth restriction

Abstract: BACKGROUND AND OBJECTIVES: To evaluate the association between unexplained or gestational-hypertension-associated fetal growth restriction (FGR) and factor V Leiden, prothrombin A20210 mutations, and methylenetetrahydrofolate reductase (MTHFR) TT 677 genotype. DESIGN AND METHODS: Sixty-one women with a previous history of FGR and 93 parous women with uneventful pregnancies from the same ethnic background were investigated for the presence of factor V (FV) Leiden, prothrombin A20210 mutations, and MTHFR TT 677 genotype. Moreover, antiphospholipid antibodies, antithrombin, protein C, and total and free protein S antigen were determined in all patients. RESULTS: Among the controls, 2 (2.2%) carried the FV Leiden mutation, 19 (20.4%) were TT MTHFR homozygotes and 1 (1.6%) carried the prothrombin A20210 allele. The FV Leiden mutation was present in 8 women with FGR (13.1%, OR: 6.9, 95%CI 1.4-33.5), the TT MTHFR homozygosity in 17 (27.8%, OR: 1.5, 95%CI 0.7-3.2) and the A20210 prothrombin allele in 7 (11.5%, OR: 5.9, 95%CI 1.2-29.4). In six cases (9.8%) there was coexistence of more than one mutation (2 had the FV Leiden and the TT MTHFR genotype and 4 carried the A20210 prothrombin allele and TT MTHFR genotype). A logistic regression analysis showed that FV Leiden and A20210 prothrombin mutations were independently associated with the occurrence of FGR. INTERPRETATION AND CONCLUSIONS: Present data indicate an association between prothrombotic genetic factors and FGR.

Connexin 31 (GJB3) is expressed in the peripheral and auditory nerves and causes neuropathy and hearing impairment

Abstract: Mutations in the connexin 31 (GJB3) gene have been found in subjects with dominant and recessive deafness and in patients with erythrokeratodermia variabilis We report here a dominant mutation in the GJB3 gene (D66del) in a family affected with peripheral neuropathy and sensorineural hearing impairment A wide range of disease severity for peripheral neuropathy, from asymptomatic cases to subjects with chronic skin ulcers in their feet and osteomyelitis leading to amputations, was detected in D66del patients Mild, often asymmetrical, hearing impairment was found in all but one patient with mutation D66del of this family and the same mutation was present in an independent family ascertained because of hearing impairment We have found mouse connexin 31 (Gjb3) gene expression in the cochlea and in the auditory and sciatic nerves, showing a pattern similar to that of Gjb1 (connexin 32), of which the human ortholog (GJB1) is involved in X-linked peripheral neuropathy This expression pattern, together with auditory-evoked brainstem anomalous response in D66del patients, indicates that hearing impairment due to GJB3 mutations involves alterations in both the cochlea and the auditory nerve Peripheral neuropathy is the third phenotypic alteration linked to GJB3 mutations, which enlarges the list of genes that cause this group of heterogeneous disorders

Predictors of failure of Helicobacter pylori eradication with the standard ‘Maastricht triple therapy’

Abstract: Background: Triple therapy with proton pump inhibitor, clarithromycin and amoxicillin has recently been proposed in Maastricht as first-line treatment for H. pylori infection. Aim: To determine predictors of unsuccessful eradication. Methods: Two hundred and forty-eight patients underwent endoscopy with biopsies for rapid urease test, histology and culture with antibiotic susceptibility tests, and 13C-UBT. All infected patients were given pantoprazole (40 mg b.d.), clarithromycin (500 mg b.d.) and amoxicillin (1 g b.d.) for 1 week. Eradication was assessed by UBT at 4–6 weeks after therapy. Results: One hundred and sixty-two of 248 patients (65%) were infected. Culture was positive in 144 (89%). Prevalence rates of metronidazole, clarithromycin and amoxicillin resistance were 14, 8 and 3%, respectively. Eradication rates (95% CI) were 63% (54.7–70.6) by intention-to-treat analysis and 67% (59.4–75.4) by per protocol analysis. Drug compliance was excellent and side-effects were mild. Age ≥ 45 years (OR: 2.35, CI: 1.30–4.25), smoking (OR: 1.37, CI 1.01–1.87) and high pre-treatment UBT results (OR: 1.36, CI: 1.08–1.72) were independent predictors of eradication failure. Gender, endoscopic findings, alcohol intake, and clarithromycin and amoxicillin resistance did not predict treatment failure. Conclusion: Despite the low prevalence of primary antibiotic resistance in our geographical area, triple therapy with pantoprazole, amoxicillin and clarithromycin achieves low eradication rates. Smoking, age and pre-treatment UBT results are predictors of potential eradication failure.

WBSCR14, a gene mapping to the Williams--Beuren syndrome deleted region, is a new member of the Mlx transcription factor network.

Abstract: Williams–Beuren syndrome (WBS) is a developmental disorder associated with haploinsufficiency of multiple genes at 7q11.23. Here, we report the functional characterization of WBS critical region gene 14 (WBSCR14), a gene contained in the WBS commonly deleted region. It encodes a basic-helix– loop–helix leucine zipper (bHLHZip) transcription factor of the Myc/Max/Mad superfamily. WBSCR14 is expressed in multiple tissues, including regions of the brain and the intestinal tract. WBSCR14 forms heterodimers with the bHLHZip protein Mlx to bind the DNA sequence CACGTG. Like Max, Mlx has no intrinsic transcriptional activity, but its association with Mad1, Mad4, Mnt or WBSCR14 can repress E-boxdependent transcription. Preliminary results suggest a possible role of WBSCR14 in growth control. Our data support the view that the Max-like bHLHZip protein, Mlx, is a key element of a transcription factor network. We thus suggest that WBSCR14 may contribute to some aspects of the WBS pathology.

Descending spinal cord volleys evoked by transcranial magnetic and electrical stimulation of the motor cortex leg area in conscious humans

Abstract: Descending corticospinal volleys evoked after transcranial magnetic or electrical stimulation of the leg area of the motor cortex were recorded from an electrode in the spinal epidural space of six conscious patients who had electrodes implanted for treatment of chronic pain, and from one anaesthetised patient undergoing surgery for a spinal tumour. At threshold, the shortest-latency volley (L1 volley) was evoked by stimulation with an anode 2 cm lateral to the vertex. Anodal stimulation at the vertex also elicited a volley at this latency in two patients, but in the other patients the first volley evoked appeared 1—1.3 ms later (L2 volley), at the same latency as the initial volley evoked by magnetic stimulation. High-intensity stimulation of any type could evoke both the L1 and L2 waves as well as later ones (L3, L4, etc.) that had a periodicity of about 1.5 ms. Voluntary contraction increased the amplitude of the L2 and later volleys, but had no effect on the L1 volley. Intracortical inhibition between pairs of magnetic stimuli resulted in clear suppression of the L4 and later waves. The L2 and L3 waves were unaffected. In the anaesthetised patient the L1 volley occurred 1.7 ms later than the volley produced by transmastoid stimulation of the corticospinal pathways in the brainstem. The L1 volley is likely to be a D wave produced by the direct activation of pyramidal axons in the subcortical white matter; the L2 and later volleys are likely to be I waves produced by the trans-synaptic activation of corticospinal neurones. The implication is that electrical stimulation with an anode at the vertex is more likely to evoke I waves preferentially than stimulation over the hand area. A more secure way to ensure D wave activation of corticospinal fibres from the leg area is to place the anode 2 cm lateral to the vertex. Transcranial magnetic stimulation (TMS) can readily activate the output of the motor cortex and evoke EMG responses in muscles throughout the body. At least for the hand area, there is now a relatively well-accepted model of how stimulation evokes the corticospinal output. At low intensities, TMS activates corticospinal neurones trans-synaptically (I waves: Day et al. 1989; Burke et al. 1993; Kaneko et al. 1996; Nakamura et al. 1996; Di Lazzaro et al. 1998a, b). Direct activation of the axon only occurs at high stimulation intensities, or if the magnetic stimulus induces current to flow in a latero-medial direction along the central sulcus (e.g. Werhahn et al. 1994; Kaneko et al. 1996; Nakamura et al. 1996; Di Lazzaro et al. 1998a). Transcranial electrical stimulation (particularly anodal stimulation; Day et al. 1989) appears to stimulate preferentially the axons of pyramidal tract cells in the subcortical white matter (D waves), and activates pyramidal neurones trans-synaptically only at higher intensities. Burke et al. (1993), who recorded descending volleys in anaesthetised patients, also noted that high-intensity anodal stimulation over the hand area could spread deep into the brain and activate corticospinal axons at two preferred points that were thought to correspond to the cerebral peduncle (the volley was 0.9 ms earlier than that evoked at threshold; termed the D2 wave), and the pyramidal decussation (1.7 ms earlier; see also Rothwell et al. (1994); termed the D3 wave). Previous studies using surface and single-unit EMG recording suggest that magnetic stimulation over the leg area recruits neurones in a subtly different way to those recruited by magnetic stimulation over the hand area. Priori et al. (1993) found that the latency of surface and single-unit EMG responses in the tibialis anterior (TA) muscle was the same for both vertex electrical and magnetic stimulation of the leg area. They assumed that electrical stimulation activated corticospinal axons in the subcortical white matter, and therefore they proposed that magnetic stimulation activated the same point. Effectively, they suggested that TMS of the leg area was much more likely to evoke a D wave than after stimulation over the hand area. Nielsen et al. (1995) also found that the latency of EMG responses in leg muscles was the same after electrical or magnetic stimulation at the vertex. However, there were two additional features of their results that led them to propose that excitation occurred at the initial segment of the corticospinal neurone rather than at the membrane of the subcortical axon. First, they used H-reflex testing to demonstrate that voluntary contraction could decrease the threshold and increase the size of descending volleys evoked by both forms of stimulation (Nielsen et al. 1993). This would be a natural consequence of stimulation at the initial segment, but not of stimulation of the axon. The second point was that if the anodal electrical stimulus was moved 2 cm lateral to the vertex, EMG latencies jumped 1-2 ms earlier still. They proposed that with this electrode position, the point of activation moved into the deep subcortical white matter like the D2 wave of Burke et al. (1993). A recent paper by Terao et al. (2000) has questioned the generality of these findings. First of all, they could not reproduce the finding of Nielsen et al. (1995) that responses to anodal electrical stimulation were earlier when the anode was placed 2 cm lateral to the vertex. Second, they compared the latency of their responses with those elicited by transmastoid electrical stimulation (Ugawa et al. 1991), which is thought to activate corticospinal fibres at the pyramidal decussation. Terao et al. (2000) found that the EMG responses evoked by lateral electrical stimulation had a latency that was 1.6-2 ms longer than those evoked by transmastoid stimulation, leading them to conclude that scalp electrical stimulation over the leg area activated corticospinal axons in the subcortical white matter (see Rothwell et al. 1994). They also found that magnetic stimulation, especially around threshold intensities, preferentially evoked EMG responses that were later than those evoked after anodal stimulation, whilst at higher intensities the latencies became equal. They argued that stimulation of the leg area was fundamentally the same as stimulation of the hand area: magnetic stimuli tended to evoke I waves, whereas vertex electrical stimulation preferentially evoked conventional D waves. These previous reports were limited by the fact that none of the authors had actually recorded the descending volleys evoked in the corticospinal tract. They had inferred what these volleys were likely to have been by examining the form of the EMG responses. One previous study (Houlden et al. 1999) has tackled specifically the recruitment of corticospinal volleys from the leg area using direct recording of descending corticospinal volleys from the epidural space of awake human subjects. They found that multiple volleys could be elicited by TMS, but that the earliest was evoked preferentially at high threshold. On the basis of the effect of anaesthesia on each volley, they identified the earliest volley as a D wave and the later volleys as I waves. They therefore favoured the original explanation of Priori et al. (1993) that magnetic stimulation could activate the axons of corticospinal neurones directly (D activation) as well as trans-synaptically (I activation). Unfortunately, they did not compare the responses to those evoked by electrical stimulation, so that the relationship between their results and those of other studies is still debateable. In particular, they did not test the hypothesis of Nielsen et al. (1995), who proposed that the first volley evoked at low threshold is initiated at the initial segment of corticospinal neurones. In the present paper we have also recorded descending volleys from the epidural space of awake human subjects following both electrical and magnetic methods of transcranial stimulation. We asked whether magnetic stimulation could evoke volleys with latencies as short as those evoked by anodal stimulation, and measured whether the size of the waves was affected by strong voluntary contraction. We also performed two other tests using double-pulse stimulation to check whether descending volleys are affected by conditioning stimulation in the same way as described for the hand area. Finally, we compared the descending volleys evoked by lateral anodal stimulation with those evoked after transmastoid stimulation.

Ultrasound-guided fine needle biopsy of the spleen: high clinical efficacy and low risk in a multicenter Italian study.

Abstract: The aim of this study was to evaluate the clinical efficacy and safety of the ultrasound-guided fine needle biopsy (UG-FNB) of the spleen in a large population of patients. We collected retrospectively the findings concerning the application of UG-FNB of the spleen from eight Italian clinical centers that utilized this technique for at least ten years. A data schedule was sent to all centers to collect information about techniques, results, and complications of UG-FNB of the spleen. We analyzed 398 biopsy procedures both on focal lesions (257 cases) and on splenic parenchyma (141 cases). The overall accuracy was 90.9% for the series as a whole, 84.9% for cytological sampling, 88.3% for microhistological sampling, and 90.3% for both cytological and histological sampling (double biopsy). Tissue core biopsy yielded better overall accuracy in patients with suspected splenic involvement by lymphoma (90.9% vs. 68.5% for cytology). The complication rate was low (no death cases, less than 1% for major complications, and 5.2% for all complications). No predictive factors were able to detect high-risk situations. The operator's skill (higher number of performed procedures) was significantly related to better overall accuracy. Conversely, the complication rate was not affected. UG-FNB of the spleen is a very effective diagnostic procedure with low risk for the patient. Aspiration cytology and core needle biopsy showed similar diagnostic yields, except for the diagnosis of splenic lymphoma, in which core needle biopsy obtained better results.

How to prolong postoperative analgesia after caudal anaesthesia with ropivacaine in children: S-ketamine versus clonidine.

Abstract: BACKGROUND The aim of the study was to determine whether caudal S-ketamine or clonidine prolonged analgesia together with ropivacaine. METHODS Sixty-three boys, aged 1-5 years, who were undergoing minor surgery, were allocated in order to receive one of three solutions for caudal anaesthesia. Group R received 2 mg x kg(-1) 0.2% ropivacaine; group C, 2 mg x kg(-1) 0.2% ropivacaine + clonidine 2 microg x kg(-1); and group K, 2 mg x kg(-1) 0.2% ropivacaine + S-ketamine 0.5 mg x kg(-1). RESULTS Postoperative analgesia assessed by CHEOPS lasted 701 min in group K (P < 0.05) compared with 492 min in group C and 291 min in group R. There were no significant differences between the groups for incidence of haemodynamic and respiratory alterations, motor block or sedation. CONCLUSIONS This study demonstrates that S-ketamine 0.5 mg x kg(-1) when added to 0.2% caudal ropivacaine provides better postoperative analgesia than clonidine without any clinically significant side-effect.

Fibroblast growth factor-II gene therapy reverts the clinical course and the pathological signs of chronic experimental autoimmune encephalomyelitis in C57BL/6 mice.

Abstract: The development of therapies aimed to promote remyelination is a major issue in chronic inflammatory demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis (MS), where the permanent neurological impairment is due to the axonal loss resulting from recurrent episodes of immune-mediated demyelination. Here, we show that the intrathecal injection of a herpes simplex virus (HSV) type-1 replication-defective multigene vector, engineered with the human fibroblast growth factor (FGF)-II gene (TH:bFGF vector), was able to significantly revert in C57BL/6 mice the clinicopathological signs of chronic experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The treatment with the TH:bFGF vector was initiated within 1 week after the clinical onset of EAE and was effective throughout the whole follow-up period (ie 60 days). The disease-ameliorating effect in FGF-II-treated mice was associated with: (1) CNS production of FGF-II from vector-infected cells which were exclusively located around the CSF space (ependymal, choroidal and leptomeningeal cells); (2) significant decrease (P < 0.01) of the number of myelinotoxic cells (T cells and macrophages) both in the CNS parenchyma and in the leptomeningeal space; and (3) significant increase (P < 0.01) of the number of oligodendrocyte precursors and of myelin-forming oligodendrocytes in areas of demyelination and axonal loss. Our results indicate that CNS gene therapy using HSV-1-derived vector coding for neurotrophic factors (ie FGF-II) is a safe and non-toxic approach that might represent a potential useful 'alternative' tool for the future treatment of immune-mediated demyelinating diseases.

Safety and efficacy of thalidomide in patients with myelofibrosis with myeloid metaplasia.

Abstract: We administered the anti-angiogenic drug thalidomide to 21 patients (12 men) with myelofibrosis with myeloid metaplasia (MMM), who were not responsive to standard treatment. Patients received thalidomide at an escalating dose from 100 to 400 mg/d. Administration of the drug was discontinued before the planned 6 months of treatment in 19 patients (90.5%), mainly because of somnolence and/or fatigue, neurological symptoms or neutropenia. Of the 13 evaluable patients (who received more than 30 d of therapy), anaemia improved in three out of seven (43%) who were treated because of anaemia; thrombocytopenia improved in two out of three (66.6%) who were treated because of thrombocytopenia; splenomegaly was reduced in four (30.8%). Undesired increases in white blood cell and platelet counts were observed in three (23.1%) and five (38.5%) patients respectively. A severity score, indexed on haematological and clinical parameters, improved in two patients (15.4%), but worsened in five (38.5%). In conclusion, standard-dose thalidomide in MMM patients is burdened with a high rate of side-effects, which prevent prolonged treatment. Because the drug is effective in improving anaemia and thrombocytopenia and in reducing splenomegaly, low-dose therapy warrants evaluation. The unexpected observation of leucocytosis and thrombocytosis suggests biological studies and better criteria for selection of patients for treatment.

Existence of a genetic risk factor on chromosome 5q in Italian coeliac disease families.

Abstract: Coeliac disease (CD) is a malabsorptive disorder of the small intestine resulting from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the whole genetic susceptibility. Several regions of potential linkage on chromosomes 3q, 5q, 10q, 11q, 15q and 19q have already been reported in the literature. These six regions were analyzed with the Maximum Lod Score method on a dense set of markers. A new sample of 89 Italian sibpairs was available for study. There was no evidence for linkage for any of the regions tested, except for chromosome 5q. For this region, our data, as well as a sample of 93 sibpairs from our first genome screen (Greco et al. 1998), are compatible with the presence of a risk factor for CD with a moderate effect.

A somatic mutation in the 5′UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency

Abstract: Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

CD38 expression correlates with adverse biological features and predicts poor clinical outcome in B-cell chronic lymphocytic leukemia.

Abstract: CD38 identifies a surface molecule with multi-functional activity. Its prognostic importance in B-cell chronic lymphocytic leukemia (B-CLL) is currently under investigation in view of the fact that two different groups have recently indicated that CD38 expression could be an independent prognostic marker in B-CLL.We analyzed the clinico-biological features of 61 immunologically typical (CD5+CD23+) B-CLL patients stratified according to the CD38 expression. Twenty-two (36%) patients expressed CD38 in more than 30% of CD19-positive cells and were considered as CD38-positive B-CLL. Atypical morphology (p 0.02), peripheral blood lymphocytosis (p 0.01) and diffuse histopathologic bone marrow pattern (p 0.003) were findings found to be closely associated with CD38 expression. On the other hand, A and B Binet stages (p 0.02) and interstitial bone marrow involvement (p 0.005) were more represented in the CD38-negative B-CLL group. Trisomy 12 was detected more frequently in the CD38-positive B-CLL group, while 13q...

Effect of age and single versus multiple dose pharmacokinetics of letrozole (Femara) in breast cancer patients.

Abstract: Letrozole (trademark Femara) is a new orally active, potent and selective aromatase inhibitor for the hormonal treatment of advanced breast cancer in postmenopausal women. The pharmacokinetics of letrozole and the suppression of peripheral estrogens were studied in 28 breast cancer patients after a single dose and at steady state. The pharmacokinetics of two distinct age groups (> or =50, or =70 years old, N=9) were compared. There were no significant differences in area under the curve (AUC) or terminal half-life between the two age groups neither after a single dose nor at steady state. However, when comparing steady state to single dose kinetics, half-life and AUC increased significantly by 42% (90% CI: 1.13, 1.78) and 28% (90% CI: 1.12, 1.47), respectively. This deviation from linearity was probably due to a partial saturation or auto-inhibition of the dominant metabolic clearance mechanism of letrozole. At steady state, approximately 70% of the administered dose was excreted in urine as unchanged letrozole (6.0+/-3.8%) or as the glucuronide of the major, pharmacologically inactive metabolite CGP44645 (64.2+/-22.7%). A single dose of letrozole caused suppression of serum estrogen levels close to the quantification limit of the assay. No difference between single dose suppression and suppression at steady state could be detected.

The dose-response relationship for clonidine added to a postoperative continuous epidural infusion of ropivacaine in children.

Abstract: UNLABELLED Epidurally administered clonidine enhances the quality and duration of postoperative analgesia when it is used as an adjunct to local anesthetics in children. We investigated the dose-response relationship for epidural clonidine when added to a continuous postoperative epidural infusion of ropivacaine. By use of an observer-blinded design, 55 pediatric patients (1-4 yr old) were randomly given a postoperative epidural infusion of plain ropivacaine 0.1% 0.2 mg. kg(-1). h(-1) (Group R), ropivacaine 0.08% 0.16 mg. kg(-1). h(-1) plus clonidine 0.04 microg. kg(-1). h(-1) (Group RC1), ropivacaine 0.08% 0.16 mg. kg(-1). h(-1) plus clonidine 0.08 microg. kg(-1). h(-1) (Group RC2), or ropivacaine 0.08% 0.16 mg. kg(-1). h(-1) plus clonidine 0.12 microg. kg(-1). h(-1) (Group RC3). A clear dose-response relationship could be identified for a continuous infusion of epidural clonidine, with clonidine dosages in the 0.08-0.12 microg. kg(-1). h(-1) range providing improved postoperative analgesia (reduced Children's Hospital of Eastern Ontario pain score, increased time to first supplemental analgesic demand, and a reduced total number of doses of supplemental analgesics during the first 48 h after surgery). Analgesia was improved without any signs of increased sedation or other side effects. The adjunct use of epidural clonidine in the dosage range of 0.08-0.12 microg. kg(-1). h(-1) appears effective and safe for use in children. IMPLICATIONS The addition of clonidine (0.08-0.12 microg.kg(-1).h(-1))to a continuous epidural infusion of ropivacaine was found to improve postoperative pain relief in children. No clinically significant signs of sedation or other side effects were observed.

Benign spindle cell tumors of the mammary stroma: diagnostic criteria, classification, and histogenesis.

Abstract: Purely benign mesenchymal spindle cell neoplasms of the breast are currently labeled under various terms in the literature (benign spindle cell tumor, fibroma, spindle cell lipoma, myofibroblastoma, solitary fibrous tumor, myogenic stromal tumor). The lack of strict diagnostic criteria to clearly indicate such mesenchymal neoplasms is the main reason which generated the risk of terming the same lesion under different names or, conversely, of collecting different types under the same term. Although such neoplasms exhibit morphological and immunophenotypical heterogeneity, they actually represent variations of the same tumor entity, likely arising from the uncommitted vimentin+/CD34+ fibroblasts of the mammary stroma, capable of multidirectional mesenchymal differentiation. To cover the entire spectrum of such lesions, the term "benign spindle cell tumors (BSCTs) of the mammary stroma" is advocated. BSCTs can be subtyped into four main groups by light microscopy (LM) and immunocytochemistry (ICC): fibroblastic, myofibroblastic, fibrohistiocytic, and mixed forms. A simple and practical approach to a nosologically correct diagnosis and a list of differential diagnoses are presented. The awareness of the diversity of morphological and immunophenotypical features of BSCTs of the mammary stroma, including uncommon variants, is helpful to avoid confusion with other monomorphic bland-looking benign and malignant spindle cell tumors and tumor-like lesions of the breast.

Prognostic factors for survival in patients with compensated cirrhosis and small hepatocellular carcinoma after percutaneous ethanol injection therapy.

Abstract: BACKGROUND The objective of this study was to identify clinical, biochemical, ultrasound, and/or pathologic parameters capable of predicting survival in a cohort of patients with well compensated cirrhosis and small hepatocellular carcinoma (HCC) who were treated with percutaneous ethanol injection (PEI). METHODS The study group included 111 patients with Child–Pugh Class A cirrhosis and with one (93 patients) or two (18 patients) HCC nodules measuring < 5 cm in greatest dimension. All patients underwent multisession PEI. The prognostic values of pretreatment and post-treatment variables were analyzed using the Kaplan–Meier method. RESULTS The overall 3-year and 5-year survival rates of 62% and 41%, respectively, were not influenced by age, gender, duration of chronic hepatitis, serum albumin, prothrombin time ratio, total bilirubin, γ-glutamyl transferase, hepatitis B surface antigen, antihepatitis C virus, HCC size, HCC ultrasound pattern, HCC histologic or cytologic grading, greatest spleen dimension, esophageal varices, or ascites. Levels of α-fetoprotein (AFP) > 14 ng/mL (P 75 IU/L (P 80 IU/L (P 13.3 ng/mL (P < 0.003) and HCC recurrence in another segment of the liver (P < 0.04) were linked to decreased survival in univariate analysis. CONCLUSIONS Among patients with Child–Pugh Class A cirrhosis with small uninodular or binodular HCC who are treated with multisession PEI, those with elevated serum AFP and transaminase levels and low platelet count before treatment are characterized by decreased survival. During follow-up, intrahepatic recurrence of the tumor is the main factor affecting survival. Cancer 2001;92:126–35. © 2001 American Cancer Society.

Diagnostic utility of an echo-contrast agent in patients with synovitis using power Doppler ultrasound: a preliminary study with comparison to contrast-enhanced MRI

Abstract: The purpose of this study was to first evaluate Levovist (Schering, Berlin, Germany), an echo-contrast agent, during power Doppler sonography (PDS) in patients with synovitis using asymptomatic joints as controls. Then we evaluated the accuracy of this technique against contrast-enhanced MRI. Forty patients (19 men and 21 women; mean age 40 years) were enrolled on the basis of clinical signs, laboratory tests, and radiographic findings positive for articular inflammatory disease. They were examined with conventional ultrasonography (US) and PDS techniques before and after intravenous contrast medium injection. Fourteen patients then underwent MRI with and without contrast medium 8–14 days after PDS studies. Three expert readers independently evaluated each examination. After contrast medium, synovium in inflammatory arthritis enhanced on PDS compared with normal joints in the same patient. Power Doppler sonography after contrast medium and MRI were concordant in all cases. Power Doppler sonography with contrast medium showed a qualitative increase in signal from synovial vessels, the first sign of synovial changes in inflammatory diseases.