Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected... more Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected females are extremely rare, mostly due to skewed X chromosome inactivation. A few papers outline MPS-II brain magnetic resonance imaging (MRI) "gestalt" in males, but neuroradiological reports on females are still lacking. We present an 11-year-old girl affected by the severe form of MPS-II who was followed up over a time span of 8 years, focusing on clinical and brain MRI evolution. In the last 2.5 years, the patient has been treated with enzyme replacement therapy (ERT) with idursulfase (Elaprase™, Shire Human Genetic Therapies AB, Sweden). On brain and cervical MRI examination, abnormalities in our patient did not differ from those detected in male patients: J-shaped pituitary sella, enlargement of perivascular spaces, brain atrophy, mild T2-hyperintensity in the paratrigonal white matter, diffuse platyspondylia, and mild odontoid dysplasia with odontoid cup. Brain atrophy progressed despite ERT introduction, whereas perivascular space enlargement did not change significantly before and after ERT. Cognitive impairment worsened independently from the course of white matter abnormality. Despite a profound knowledge of genetic and biochemical aspects in MPS-II, neuroradiology is still poorly characterized, especially in female patients. Spinal and brain involvement and its natural course and evolution after ERT introduction still need to be clarified. Abbreviations MPS-II Mucopolysaccharidosis type II IDS Iduronate-2-sulphatase WM White matter WMAs White matter abnormalities ERT Enzyme replacement therapy V-R Virchow-Robin
Maroteaux-Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal en... more Maroteaux-Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal enzyme, arylsulfatase B (ARSB). Among the numerous genomic lesions reported till now, the sequence variant, c.1151G4A (p.S384N), has been associated with a severe phenotype in more than 10% of the patients. We now report the first in vivo demonstration of the polymorphic nature of p.S384N, revealed during the segregation analysis in a family at risk for Maroteaux-Lamy syndrome. The proband, compound heterozygous for c.[944G4A] þ [245T4G] (p.[R315Q] þ [L82R]), did not carry the p.S384N change, which was instead present in two healthy members of the family, in trans with the causative mutations, p.R315Q and p.L82R, respectively. The hypothesis that p.S384N was a polymorphism was further addressed by reverse dot-blot analysis of 400 control alleles, estimating an allele frequency of 4.5%. To predict the consequences of p.R315Q, p.L82R and p.S384N, we also modeled and compared the three amino-acid changes in the three-dimensional ARSB structure. The in silico analysis predicted a local protein misfolding in the presence of p.R315Q and p.L82R. On the contrary, no evident problem was predicted in the case of p.S384N, occurring on the protein surface, far from the active site. Overall, these findings strongly support the hypothesis that the non-synonymous change p.S384N is a polymorphism. Moreover, our results emphasize the need for caution in drawing conclusions from a novel variant allele before screening at least 50 healthy control subjects.
Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by ... more Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.
With a view to using multiple injections of anticancer dendritic cell (DC)-based vaccines, we eva... more With a view to using multiple injections of anticancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34 1 cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34 1 cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membraneexposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-l-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP 1 DCs: 50^2%, viability 95%) under both smalland large-scale conditions. The addition of poly-l-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a 1 /AP 1 cells 85^3% and 80^2% respectively). Polycations made it possible to
Italian patients undergoing ERT, we observed that TNFα (tumour necrosis factor α) might be a biom... more Italian patients undergoing ERT, we observed that TNFα (tumour necrosis factor α) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFα expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.
Biochemical and Biophysical Research Communications, 1997
first been cloned into plasmids (3) and subsequently The bacteriophage T7 binary expression syste... more first been cloned into plasmids (3) and subsequently The bacteriophage T7 binary expression system is introduced into vaccinia virus (4), baculovirus (5) and, widely used in vitro for high level selective expression recently, adenovirus (6) vectors. However, to our of cloned genes but its application to in vivo models knowledge, the possibility to apply the T7 binary syshas not yet been investigated. In the present work, we tem in an in vivo setting has not yet been investigated.
Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected... more Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected females are extremely rare, mostly due to skewed X chromosome inactivation. A few papers outline MPS-II brain magnetic resonance imaging (MRI) "gestalt" in males, but neuroradiological reports on females are still lacking. We present an 11-year-old girl affected by the severe form of MPS-II who was followed up over a time span of 8 years, focusing on clinical and brain MRI evolution. In the last 2.5 years, the patient has been treated with enzyme replacement therapy (ERT) with idursulfase (Elaprase™, Shire Human Genetic Therapies AB, Sweden). On brain and cervical MRI examination, abnormalities in our patient did not differ from those detected in male patients: J-shaped pituitary sella, enlargement of perivascular spaces, brain atrophy, mild T2-hyperintensity in the paratrigonal white matter, diffuse platyspondylia, and mild odontoid dysplasia with odontoid cup. Brain atrophy progressed despite ERT introduction, whereas perivascular space enlargement did not change significantly before and after ERT. Cognitive impairment worsened independently from the course of white matter abnormality. Despite a profound knowledge of genetic and biochemical aspects in MPS-II, neuroradiology is still poorly characterized, especially in female patients. Spinal and brain involvement and its natural course and evolution after ERT introduction still need to be clarified.
Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected... more Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected females are extremely rare, mostly due to skewed X chromosome inactivation. A few papers outline MPS-II brain magnetic resonance imaging (MRI) "gestalt" in males, but neuroradiological reports on females are still lacking. We present an 11-year-old girl affected by the severe form of MPS-II who was followed up over a time span of 8 years, focusing on clinical and brain MRI evolution. In the last 2.5 years, the patient has been treated with enzyme replacement therapy (ERT) with idursulfase (Elaprase™, Shire Human Genetic Therapies AB, Sweden). On brain and cervical MRI examination, abnormalities in our patient did not differ from those detected in male patients: J-shaped pituitary sella, enlargement of perivascular spaces, brain atrophy, mild T2-hyperintensity in the paratrigonal white matter, diffuse platyspondylia, and mild odontoid dysplasia with odontoid cup. Brain atrophy progressed despite ERT introduction, whereas perivascular space enlargement did not change significantly before and after ERT. Cognitive impairment worsened independently from the course of white matter abnormality. Despite a profound knowledge of genetic and biochemical aspects in MPS-II, neuroradiology is still poorly characterized, especially in female patients. Spinal and brain involvement and its natural course and evolution after ERT introduction still need to be clarified. Abbreviations MPS-II Mucopolysaccharidosis type II IDS Iduronate-2-sulphatase WM White matter WMAs White matter abnormalities ERT Enzyme replacement therapy V-R Virchow-Robin
Maroteaux-Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal en... more Maroteaux-Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal enzyme, arylsulfatase B (ARSB). Among the numerous genomic lesions reported till now, the sequence variant, c.1151G4A (p.S384N), has been associated with a severe phenotype in more than 10% of the patients. We now report the first in vivo demonstration of the polymorphic nature of p.S384N, revealed during the segregation analysis in a family at risk for Maroteaux-Lamy syndrome. The proband, compound heterozygous for c.[944G4A] þ [245T4G] (p.[R315Q] þ [L82R]), did not carry the p.S384N change, which was instead present in two healthy members of the family, in trans with the causative mutations, p.R315Q and p.L82R, respectively. The hypothesis that p.S384N was a polymorphism was further addressed by reverse dot-blot analysis of 400 control alleles, estimating an allele frequency of 4.5%. To predict the consequences of p.R315Q, p.L82R and p.S384N, we also modeled and compared the three amino-acid changes in the three-dimensional ARSB structure. The in silico analysis predicted a local protein misfolding in the presence of p.R315Q and p.L82R. On the contrary, no evident problem was predicted in the case of p.S384N, occurring on the protein surface, far from the active site. Overall, these findings strongly support the hypothesis that the non-synonymous change p.S384N is a polymorphism. Moreover, our results emphasize the need for caution in drawing conclusions from a novel variant allele before screening at least 50 healthy control subjects.
Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by ... more Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.
With a view to using multiple injections of anticancer dendritic cell (DC)-based vaccines, we eva... more With a view to using multiple injections of anticancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34 1 cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34 1 cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membraneexposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-l-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP 1 DCs: 50^2%, viability 95%) under both smalland large-scale conditions. The addition of poly-l-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a 1 /AP 1 cells 85^3% and 80^2% respectively). Polycations made it possible to
Italian patients undergoing ERT, we observed that TNFα (tumour necrosis factor α) might be a biom... more Italian patients undergoing ERT, we observed that TNFα (tumour necrosis factor α) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFα expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.
Biochemical and Biophysical Research Communications, 1997
first been cloned into plasmids (3) and subsequently The bacteriophage T7 binary expression syste... more first been cloned into plasmids (3) and subsequently The bacteriophage T7 binary expression system is introduced into vaccinia virus (4), baculovirus (5) and, widely used in vitro for high level selective expression recently, adenovirus (6) vectors. However, to our of cloned genes but its application to in vivo models knowledge, the possibility to apply the T7 binary syshas not yet been investigated. In the present work, we tem in an in vivo setting has not yet been investigated.
Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected... more Mucopolysaccharidosis type II (MPS-II, Hunter disease) is a X-linked recessive disorder. Affected females are extremely rare, mostly due to skewed X chromosome inactivation. A few papers outline MPS-II brain magnetic resonance imaging (MRI) "gestalt" in males, but neuroradiological reports on females are still lacking. We present an 11-year-old girl affected by the severe form of MPS-II who was followed up over a time span of 8 years, focusing on clinical and brain MRI evolution. In the last 2.5 years, the patient has been treated with enzyme replacement therapy (ERT) with idursulfase (Elaprase™, Shire Human Genetic Therapies AB, Sweden). On brain and cervical MRI examination, abnormalities in our patient did not differ from those detected in male patients: J-shaped pituitary sella, enlargement of perivascular spaces, brain atrophy, mild T2-hyperintensity in the paratrigonal white matter, diffuse platyspondylia, and mild odontoid dysplasia with odontoid cup. Brain atrophy progressed despite ERT introduction, whereas perivascular space enlargement did not change significantly before and after ERT. Cognitive impairment worsened independently from the course of white matter abnormality. Despite a profound knowledge of genetic and biochemical aspects in MPS-II, neuroradiology is still poorly characterized, especially in female patients. Spinal and brain involvement and its natural course and evolution after ERT introduction still need to be clarified.
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Papers by Rosela Tomanin