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Biomedical subjects

M Milani

Publications and source records attributed to M Milani.

At least 37 records · Page 2Linked to original sources
PubMed2001

Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.

The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

Amino Acid Substitution↗
PubMed2001

Charcot-Marie-Tooth disease type I and related demyelinating neuropathies: Mutation analysis in a large cohort of Italian families.

Charcot-Marie-Tooth neuropathy type 1 (CMT1), the most common hereditary neurological disorder in humans, is characterized by clinical and genetic heterogeneity. It is caused mainly by a 1.5 Mb duplication in 17p11.2, but also by mutations in the myelin genes PMP22 (peripheral myelin protein 22), MPZ (myelin protein zero), Cx32 (connexin 32; also called GJB1), and EGR2 (early growth response 2). In this study, we have screened 172 index cases of Italian families in which there was at least one subject with a CMT1 diagnosis for the duplication on 17p11.2 and mutations in these genes. Among 170 informative unrelated patients, the overall duplication frequency was 57.6%. A difference could be observed between the duplication frequency in familial cases (71.6%) and that observed in non-familial cases (36.8%). Among the non-duplicated patients, 12 were mutated in Cx32, four in MPZ, two in PMP22, and none in the EGR2. In the non-duplicated cases, the overall point mutation frequency for these genes was 25.0%. We describe the mutations identified, and consider possible genotype-phenotype correlation.

Charcot-Marie-Tooth Disease↗
PubMed2001

Evaluation of the efficacy and tolerability of oral terbinafine (Daskil) in patients with seborrhoeic dermatitis. A multicentre, randomized, investigator-blinded, placebo-controlled trial.

BACKGROUND: Previous uncontrolled trials have suggested that oral terbinafine, an antimycotic allylamine compound, could be useful in the treatment of seborrhoeic dermatitis. OBJECTIVES: To investigate in a placebo-controlled trial the clinical efficacy of oral terbinafine (Daskil(R), Mipharm, Milan, Italy) in patients with moderate to severe seborrhoeic dermatitis. METHODS: Sixty outpatients (mean +/- SD age 37 +/- 11 years; 32 men and 28 women) with moderate to severe seborrhoeic dermatitis were enrolled in a multicentre, randomized, placebo-controlled, investigator-blinded, parallel-group, 12-week study. After a 2-week wash-out period, enrolled patients were randomized to treatment with oral terbinafine 250 mg daily (n = 30) or placebo (moisturizing ointment) (n = 30) applied twice daily for 4 weeks (weeks 0-4). Patients were followed up for an additional 8 weeks after completion of treatment and were clinically evaluated at weeks 0, 2, 4 and 12 by an investigator unaware of the patient's type of treatment. The primary end-point of the study was clinical evaluation of erythema, scaling and itching, each scored on a 0-3 scale. A global clinical score, representing the sum of each evaluated symptom, was also calculated. RESULTS: Demographic and clinical data were equally balanced between the placebo and terbinafine groups. All enrolled patients concluded the study. At baseline, the mean +/- SD global clinical score was 7.4 +/- 1.3 in the placebo group and 7.7 +/- 1.0 in the terbinafine-treated group. At weeks 4 and 12 the mean +/- SD global clinical score in the placebo group was 5.9 +/- 1.7 and 6.3 +/- 1.2, respectively, which was not significantly different from baseline. As compared with baseline values and the placebo group, terbinafine treatment significantly (P < 0.0001, Tukey-Kramer test) reduced the mean +/- SD global clinical score (to 1.0 +/- 1.1 at week 4, and 1.2 +/- 1.4 at week 12), as well as the individual erythema, scaling and itching scores. No serious adverse events were recorded during the study in either group. CONCLUSIONS: This is the first controlled trial that has shown oral terbinafine to be effective in the treatment of moderate to severe seborrhoeic dermatitis. Clinical improvement following 4 weeks treatment with terbinafine was maintained 8 weeks after completing treatment.

Administration, Oral↗
PubMed2001

Abnormalities of Gq-mediated cell signaling in Bartter and Gitelman syndromes.

BACKGROUND: The constitutive endothelial isoform of nitric oxide synthase (ecNOS) and nitric oxide (NO) production are increased in patients with Bartter syndrome (BS) and Gitelman (GS) syndrome and may reduce vascular tone. Moreover, these patients present an abnormal cell signaling [reduced stimulated intracellular calcium ([Ca(2+)](i)) and inositol-1,4,5,triphosphate ([IP(3)](i)) in neutrophils], suggesting the presence of a generalized reduction of protein kinase C (PKC) and cell reactivity. Since PKC regulates ecNOS gene expression, we evaluated the signal transduction system involving Gq protein, PKC, and ecNOS in circulating nucleated cells from patients with BS/GS. METHODS: Nucleated blood cells from 2 BS and 7 GS and from 10 controls (C) were used. PKC activity was evaluated in neutrophils by radioenzymatic assay; PKC alpha concentration was evaluated in monocytes by Western blot analysis. ecNOS and G alpha q mRNA production was evaluated in monocytes by reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers and quantitated by PCR-based semiquantitative analysis of ecNOS and G alpha q mRNA expression. RESULTS: Cytosol and membrane basal PKC activity were similar in neutrophils from BS/GS and C (70 +/- 3 vs. 80 +/- 2; 37 +/- 3 vs. 46 +/- 2 pmol/min/mg protein, respectively), while fMLP-stimulated membrane PKC activity increased to a lower extent in BS/GS (from 43 +/- 2 to 53 +/- 3 vs. 38 +/- 2 to 66 +/- 3 pmol/min/mg protein, P < 0.05 for the difference). Membrane PKC alpha expression was similar in basal conditions (8.5 +/- 1.5 vs. 12.4 +/- 4.0 densitometric units), but increased after fMLP was reduced in BS/GS (4.5 +/- 1.4 vs. 9.5 +/- 2.1, P < 0.01). In BS/GS, PKC stimulation with PMA dose dependently reduced ecNOS gene expression (from 0.80 +/- 0.05 to 0.78 +/- 0.03 densitometric units; PMA 50 nmol/L, P = NS; to 0.55 +/- 0.07, PMA 100 nmol/L, P < 0.001) to an undetectable expression (PMA 200 nmol/L). Qualitatively similar effects were seen in monocytes from control subjects. Incubation of monocytes from patients and controls with the PKC inhibitor GF109203X increased ecNOS mRNA, with no difference between patients and controls. G alpha q mRNA was reduced in BS/GS versus controls (0.87 +/- 0.013 vs. 0.98 +/- 0.005 densitometric units, P < 0.0004). CONCLUSION: An abnormal G alpha q expression blunts cell signaling and PKC production in BS/GS. A reduced PKC up-regulated NO system may contribute to the vascular hyporeactivity of BS/GS.

Adult↗
PubMed2001

Intrathecal chemotherapy with antineoplastic agents in children.

Intrathecal chemotherapy with antineoplastic agents is mainly utilised in children with leukaemia and lymphoma, and in selected brain tumours. In these diseases, intrathecal use is restricted to methotrexate (MTX), cytosine arabinoside (Ara-C) and corticosteroids. A number of other agents are, at the present time, under evaluation. Intrathecal MTX administered sequentially with systemic high dose MTX infusion prolongs therapeutic cerebral spinal fluid (CSF) levels of the drug. Prolonged therapeutic CSF levels can also be achieved by giving repeated small intrathecal doses of MTX over an extended period in selected patients, with an implanted Ommaya reservoir. In the CSF, the metabolic inactivation of Ara-C is significantly lower than in plasma with a CSF clearance similar to the rate of CSF bulk flow. A slow-release formulation of Ara-C may be given intrathecally, resulting in a prolonged cytotoxic concentration in the CSF. CNS relapse and neurotoxicity in patients with acute lymphoblastic leukaemia, especially younger children, may be reduced by using age-related dosing of intrathecal MTX and Ara-C. Hydrocortisone is used in combination with MTX and Ara-C for so-called 'triple intrathecal chemotherapy' in the treatment of meningeal leukaemia. Intrathecal thiotepa does not appear to be advantageous over systemic administration in patients with brain and meningeal leukaemia. Monoclonal antibodies, reactive with tumour-associated antigens, can be used as delivery systems for chemotherapeutic agents and radionuclides. However, the development of this new approach is currently under evaluation in larger clinical studies. Neurological adverse effects may be expected with intrathecal chemotherapy and are increased by high dose systemic therapy, concomitant cranial radiotherapy or meningeal infiltration by neoplastic cells. Inadvertant intrathecal administration of antineoplastic agents that are indicated for systemic administration only, is dangerous and may result in a fatal outcome.

Adrenal Cortex Hormones↗
PubMed2000

L-asparagine depletion and L-asparaginase activity in children with acute lymphoblastic leukemia receiving i.m. or i.v. Erwinia C. or E. coli L-asparaginase as first exposure.

PURPOSE: The present study was aimed at investigating L-asparaginase (L-ASE) activity (in plasma) and L-asparagine (L-ASN) depletion (in plasma and CSF) in children with newly diagnosed acute lymphoblastic leukemia (ALL) exposed for the first time to different L-ASE products. PATIENTS AND METHODS: During the induction treatment of the AIEOP ALL 95 study, 62 patients were treated with either Erwinase (n = 15), or E. coli medac (n = 47) L-ASE products, given either i.m. or i.v., at the standard dosage of 10,000 IU/m2, q 3 days x 8 (first exposure). RESULTS: Plasma and CSF L-ASN trough levels were undetectable in all cases, including those with L-ASE trough activity < 50 mU/ml. L-ASE trough activity during the administration of medac was however significantly higher when compared with that of Erwinase. CONCLUSIONS: L-ASN depletion after a first exposure to standard doses of Erwinase or medac is obtained in virtually all patients. No differences are seen between the I.M. or I.V. administration routes but the medac product is associated with a significantly higher enzyme activity in respect of Erwinase. L-ASN levels may be undetectable also in patients with L-ASE trough activity levels < 50 mU/ml, challenging the current opinion that an activity level of 100 mU/ml is needed to obtain L-ASN depletion.

Antineoplastic Agents↗
PubMed2000

Distribution of age-related thymulin titres in normal subjects through the course of life.

The thymus has a dominant immunological role in utero and in early childhood, being a primary source of T lymphopoiesis, and its investigation may be particularly relevant for the immunological study of paediatric patients. Thymulin, a nonapeptide secreted by the thymus, is an essential hormone for T lymphocyte differentiation and function. As thymulin values in the normal population have not been well documented, especially for children under the age of 1 year, we detail thymic endocrine function by presenting age-related plasma thymulin levels in a large series (n = 93) of healthy individuals, ranging from birth to old age. We demonstrate that thymulin is already detectable at birth; it then gradually increases with age, reaching the highest level in children aged 5-10 years. Starting at adolescence, thymulin titres gradually start to fall, reaching the lowest value at 36 years of age and remaining steady until 80 years (the oldest person tested).

Adolescent↗
PubMed2000

Primary thymic endocrine failure in HIV-1-infected children.

Thymulin, an essential hormone for the T lymphocyte differentiation process and function, was evaluated to asses thymic endocrine function in a cohort of 17 HIV-1-infected children aged between 2 months and 14 years, 18 seroreverted subjects and 47 normal controls. The rosette inhibition assay by Dardenne and Bach (1975) is the only method available to evaluate the biologically active form of this hormone (thymulin or Zn-facteur thymique sérique, Zn-FTS), as immunoassays cannot discriminate between thymulin and the inactive form of the hormone not containing Zn (FTS). HIV-1 patients presented undetectable or significantly lowered plasma levels of thymulin. Plasma zinc levels were significantly reduced in patients although inactive, zinc-unbound thymulin molecules were not demonstrated. The investigation of inhibitory anti-thymulin molecules performed in all patients was negative. Thymulin titers did not correlate with CD4+ lymphocyte count at the different disease stages. This study suggests that a primary thymic endocrine deficiency is present in HIV children. The critical importance of these results in assessing disease progression and a potential therapeutic approach are discussed.

Adolescent↗
PubMed2000

Immortalized luteinizing hormone-releasing hormone neurons show a different migratory activity in vitro.

The development of two cell lines (GT1 and GN) of immortalized LHRH neurons has allowed an accurate study of the mechanisms controlling the synthesis and the secretion of LHRH. These cell lines, obtained in mice by genetic targeted tumorigenesis, retain many of the phenotypic characteristics of LHRH neurons. Of interest, GT1 cells derive from an hypothalamic tumor, whereas GN cells were obtained from a tumor localized in the olfactory bulb. The different origin of these cell lines lead to hypothesize that they might represent hypothalamic postmigratory neurons (GT1 cells), or LHRH neurons blocked at an early stage of their migration (GN cells). Using different experimental procedures, we found that the two cell subclones GT1-7 and GN11 express a different morphology and migratory behavior in vitro. In particular, we found that GN11 cells, but not GT1-7 cells, show the morphological shape of migrating neurons. When analyzing the spontaneous motility we found that only GN11 cells express a high capacity of migrating in a matrix of collagen gel. Moreover, in a chemomigratory assay GN11 cells did show a significant response to the chemotactic stimulus represented by the FBS. On the contrary, GT1-7 cells show very low spontaneous motility and appear insensitive to the FBS stimulus. These results suggest that the simultaneous use of the GT1-7/GN11 cells may represent an experimental tool for screening the factors possibly involved in the control of the migratory processes of LHRH neurons in normal and in pathological conditions, such as those due to their impaired migration, like it happens in Kallmann's syndrome.

Animals↗
PubMed2000

Fluvastatin reduces soluble P-selectin and ICAM-1 levels in hypercholesterolemic patients: role of nitric oxide.

BACKGROUND: Lipid-lowering therapy with 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase inhibitors reduces the incidence of atherosclerosis-related cardiovascular events. Adhesion molecules, regulating interactions between vascular and circulating cells, may play a central role in the pathogenesis of atherosclerosis and related complications. In the present report we examined the impact of the HMG-CoA reductase inhibitor fluvastatin on plasma levels of P-selectin and ICAM-1. METHODS: Plasma levels of P-selectin and ICAM-1 were determined using an enzyme immunoassay in 26 patients with type IIa hypercholesterolemia randomized to treatment with either fluvastatin (80 mg/d) or placebo in a double blind fashion for 12 weeks. RESULTS: Fluvastatin administration reduced either P-selectin (118 +/- 63 vs 81 +/- 36 ng/mL [-31%], P = 0.0015) or ICAM-1 (264 +/- 75 vs 228 +/- 68 ng/mL [-13.7%], P = 0.0033) levels. Fluvastatin also lowered urinary 11-dehydro-TXB2 (1396 +/- 536 vs 1009 +/- 378 pg/mg creatinine [-27%], P = 0.0015) and von Willebrand Factor levels (1456 +/- 716 vs 1203 +/- 527 U/L [-17.4%], P = 0.0275), and a direct correlation was observed between P-selectin and 11-dehydro-TXB2 levels (r = 0.588, P = 0.0033). Patients treated with fluvastatin displayed an increase in nitric oxide (NO) generation, evaluated with measurements of serum NO2-/NO3-, (4.7 +/- 1 vs 8.9 +/- 3.1) mumol/L [98%], P = 0.0046). Moreover, an inverse correlation was observed between NO2-/NO3- and P-selectin (r = -0.420; P = 0.0343), 11-dehydro-TXB2 (r = -0.511; P = 0.0106), or LDL (r = -0.742; P = 0.0002) levels. CONCLUSIONS: These results may provide novel biochemical basis for the beneficial clinical effects of HMG-CoA reductase inhibitors in hypercholesterolemia.

Adult↗
PubMed1999

Photodynamic action of porphyrin on Ca2+ influx in endoplasmic reticulum: a comparison with mitochondria.

We have studied the distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) in mitochondria and endoplasmic reticulum after isolation from rat liver. The photosensitizing efficiency of porphyrin on the Ca2+ influx function of microsomes has been compared with that obtained on Ca2+ uptake in mitochondria. HP and PP are accumulated in microsomes to a greater extent than in mitochondria, both porphyrins binding to membrane protein sites. The Ca2+ influx functions of mitochondria and microsomes, before and after irradiation in the presence of HP or PP, were studied by following the changes in the free Ca2+ concentration in the medium as revealed by the variations in fluorescence intensity of the Ca2+ indicator Calcium Green-1. For the same amount of incorporated porphyrin, the Ca2+ influx function of microsomes is degraded by irradiation more rapidly than that of mitochondria. The protective effect of dithiothreitol suggests that thiol groups in the Ca2+-transporting enzyme are the preferential targets of the photodynamic effect. These results suggest that intracellular Ca2+ movements are altered primarily by the endoplasmic reticulum rather than by mitochondrial damage, in good agreement with other observations made in porphyrin-loaded irradiated cells.

Animals↗
PubMed1999

Successful treatment of intrathecal methotrexate overdose with folinic acid rescue: a case report.

A 7-y-old boy with acute lymphoblastic leukaemia (ALL) received 600 mg of i.v. methotrexate (MTX) over 2 h, followed by triple intrathecal therapy (TIT) with cytosine arabinoside 30 mg, methylprednisolone 10 mg and MTX 300 mg (instead of the prescribed 12 mg). Ninety minutes later the patient developed headache, loss of consciousness and generalized hypertonia. He was transferred to the Intensive Care Unit, intubated and treated with phenobarbital. Three hours after the TIT, the levogyrus form of folinic acid (equivalent to double doses of the racemic product) was started i.v. at a dose of 100 mg every 3 h for 24 h, and every 6 h in the following 24 h. Cerebrospinal fluid was examined and was found normal. The patient subsequently remained in normal neurological status. The favourable outcome in our case suggests that folinic acid rescue may be adequate to prevent sequelae in patients who undergo intrathecal MTX overdoses up to 300 mg.

Antidotes↗
PubMed1999

Oral administration of an immunodominant T-cell epitope downregulates Th1/Th2 cytokines and prevents experimental myasthenia gravis.

The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell-dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell-dependent disease. We report that oral administration of the T-cell epitope alpha146-162 of the Torpedo californica acetylcholine receptor (TAChR) alpha-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Ab's to mouse AChR and reduced AChR loss in muscle. The effect of Talpha146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Talpha146-162 was mediated by reduced production of IFN-gamma, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-beta-secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease.

Administration, Oral↗
PubMed1999

Acute leukaemia immunophenotyping in bone-marrow routine sections.

Immunohistochemistry of acute leukaemias in bone-marrow paraffin sections is commonly thought to be useless because of the poor preservation of many lineage-related markers. The recent development of antibodies against fixative-resistant epitopes and of new antigen retrieval techniques, however, has expanded the possibility of accurately testing routine samples. To assess the relevance of paraffin section phenotyping in lineage determination, 110 examples of acute leukaemia were studied by specific antibodies against CD1a, CD3, CD15, CD20, CD34, CD68, CD79a, TdT, myeloperoxidase, glycophorin A, and factor-VIII-related antigen. The cases included 59 acute myeloid leukaemias, classified according to the FAB cooperative group criteria, 39 precursor B-cell acute lymphoblastic leukaemias (ALLs), seven T-ALLs, and five mixed precursor B-cell/myeloid acute leukaemias. The combination of the markers employed always allowed the identification of the cell lineage (myeloid, lymphoid or mixed) and, in some instances, of phenotypic profiles characteristic of distinct acute leukaemia subtypes. According to the results obtained, bone-marrow biopsy may be regarded as a reliable tool for acute leukaemia diagnosis; this observation is of practical relevance especially for the classification of cases which lack circulating blasts in the peripheral blood or showing dry tap at bone-marrow aspiration.

Acute Disease↗
PubMed1999

Effects of aspirin or picotamide, an antithromboxane agent, in combination with low-intensity oral anticoagulation in patients with acute myocardial infarction: a controlled randomized pilot trial.

BACKGROUND AND OBJECTIVE: Combined treatment with antiplatelet drugs and oral anticoagulants seems more effective than monotherapy in the reduction of thrombotic episodes in patients with ischemic heart diseases. We compared the safety and efficacy of two different antiplatelet drugs, aspirin (asa) and picotamide (pico)--a dual antithromboxane agent--in combination with low-intensity oral anticoagulation with warfarin or acenocoumarol in acute myocardial infarction (AMI). PATIENTS AND METHODS: Primary endpoint of the study was to compare the incidence of major events (death, reinfarction, postinfarction angina and heart failure) in AMI patients undergoing thrombolytic therapy. In a controlled randomized parallel group pilot study, 101 patients with AMI were enrolled and treated with asa 160 mg/die plus low-intensity oral anticoagulation (target INR: 1.5-2.5) (n = 51) or pico 300 mg/tid plus low-intensity oral anticoagulation (n = 50). Secondary endpoint of the study was to compare the cumulative incidence of major events plus major hemorrhagic episodes defined as macroscopic hematuria, nose bleeding and melena. RESULTS: The two groups were well matched regarding the main demographic and clinical variables. AMI location was anterior in 22 and 20 patients in the pico and asa group respectively, inferoposterior in 27 (pico) and 29 (asa) patients. At the end of the six-month period, major events were observed in 20 patients in the pico group and in 31 patients in the asa group (p < 0.05). The cumulative incidence of major clinical events plus major hemorrhagic episodes was significantly lower in the pico group in comparison with the asa group (28 vs 48; p < 0.001). CONCLUSION: The results of this pilot study suggest that combination therapy with picotamide and low-intensity oral anticoagulation could be a safe and effective alternative to aspirin in patient with AMI. This hypothesis should be confirmed by controlled randomized trial with an adequate sample size.

Administration, Oral↗