The role of unidentified bright objects in the neurocognitive profile of neurofibromatosis type 1 children: a volumetric MRI analysis.

PURPOSE: Cognitive impairment is described in 80% of Neurofibromatosis type 1 (NF1) patients. Brain focal areas of T2w increased signal intensity on MRI, the so-called Unidentified Bright Objects (UBOs) have been hypothesized to be related to cognitive dysfunction, although conflicting results are available in literature. Here, we investigated the possible relation between UBOs' volume, cognitive impairment, and language disability in NF1 patients. MATERIAL AND METHODS: In this retrospective study, clinical and MRI data of 21 NF1 patients (M/F = 12/9; mean age 10.1 ± 4.5) were evaluated. Brain intellectual functioning and language abilities were assessed with specific scales, while the analyzed MRI sequences included axial 2D-T2-weighted and FLAIR sequences. These images were used independently for UBOs segmentation with a semiautomatic approach and obtained volumes were normalized for biparietal diameters to take into account for brain volume. Possible differences in terms of normalized UBOs volumes were probed between cognitively affected and preserved patients, as well as between subjects with or without language impairment. RESULTS: Patients cognitively affected were not different in terms of UBOs volume compared to those preserved (p = 0.35 and p = 0.30, for T2-weighted and FLAIR images, respectively). Similarly, no differences were found between patients with and without language impairment (p = 0.47 and p = 0.40, for the two sequences). CONCLUSIONS: The relation between UBOs and cognition in children with NF1 has been already investigated in literature, although leading to conflicting results. Our study expands the current knowledge, showing a lack of correlation between UBOs volume and both cognitive impairment and language disability in NF1 patients.

Obstructive Sleep Apnoea in Children with Down Syndrome: A Multidisciplinary Approach.

A comprehensive evaluation of obstructive sleep apnoea (OSA) may allow for the development of more efficient management of Down syndrome (DS). We aimed to evaluate the effect of a multidisciplinary approach to DS with OSA. A total of 48 DS children aged 4−12 years were prospectively investigated with nasal endoscopy, orthodontic examination, and overnight polygraphy (PG); the Italian Child Sleep Habits Questionnaire (CSHQ-IT) was filled out by the mothers. The total CSHQ-IT score was 63 (96% of children reporting sleep problems). The major ear, nose, and throat characteristics were enlarged palatine tonsils (62%), adenoid tonsils (85%), and chronic rhinosinusitis (85%). DS children showed orthognathic profile in 68% of cases, class I relationship in 63%, and cross-bite in 51%. PG revealed OSA in 67% of cases (37% mild, 63% moderate−severe). The oxygen desaturation index (ODI) was higher in the group with OSA (5.2) than with non-OSA (1.3; p < 0.001). The ODI was higher (p = 0.001) and SpO2 lower (p = 0.03) in children with moderate−severe OSA than with mild OSA. The apnoea−hypopnea index (AHI) and percentage time with SpO2 < 90% were higher in DS children with grade III than with grade I or II adenoids (5 vs. 1, p = 0.04, and 1.2 vs. 0.1, p = 0.01, respectively). No significant correlations were found between PG and the total CSHQ-IT score or orthodontic data. However, children showing associated cross-bite, grade III adenoids and size 3 or 4 palatine tonsils showed higher AHI and ODI than those without (p = 0.01 and p = 0.04, respectively). A coordinated multidisciplinary approach with overnight PG is a valuable tool when developing diagnostic protocols for OSA in DS.

Prospective evaluation of autoimmune and non-autoimmune subclinical hypothyroidism in Down syndrome children.

OBJECTIVE: To evaluate the prevalence and natural course of autoimmune and non-autoimmune subclinical hypothyroidism (SH) in Down syndrome (DS) children and adolescents. DESIGN: Prospective multicenter study. METHODS: For the study, 101 DS patients with SH (TSH 5-10 mIU/L; FT4 12-22 pmol/L), aged 2-17 years at SH diagnosis were enrolled. Annual monitoring of TSH, FT4, BMI, height, and L-thyroxine dose was recorded for 5 years. Thyroid autoimmunity was tested at diagnosis and at the end of follow-up. RESULTS: Thirty-seven out of 101 patients displayed autoantibody positivity (group A); the remaining 64 were classified as non-autoimmune SH (group B). Group A was characterized by higher median age at SH diagnosis and by more frequent family history of thyroid disease (6.6 vs 4.7 years, P = 0.001; 32.4% vs 7.8%, P = 0.001 respectively), whereas congenital heart defects were more common in group B (65.6% vs 43.2%, P = 0.028). Gender, median BMI (SDS), height (SDS), FT4, and TSH were similar in both groups. At the end of follow-up: 35.1% of group A patients developed overt hypothyroidism (OH) vs 17.2% of group B (P = 0.041); 31.25% of group B vs 10.8% of group A became biochemically euthyroid (P = 0.02); and 37.8% of group A vs 51.5% of group B still had SH condition (P = 0.183). Logistic regression suggested autoimmunity (OR = 3.2) and baseline TSH values (OR = 1.13) as predictive factors of the evolution from SH to OH. CONCLUSIONS: In DS children, non-autoimmune SH showed higher prevalence and earlier onset. The risk of thyroid function deterioration over time seems to be influenced by thyroid autoimmunity and higher baseline TSH values.

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.

B-cell receptor-guided delivery of peptide-siRNA complex for B-cell lymphoma therapy.

BACKGROUND: Despite the clinical response of conventional anticancer therapy, including chemotherapeutic treatments, radiation therapy and corticosteroids, tumorigenic B-cell lymphomas show an incomplete response to clinical practices that result in a minimal residual disease (MRD) where few residual neoplastic cells undetected in vivo, replenish the cancer cell reservoir. This scenario, which is also shared with other cancer diseases, requires the development of strategies to advance in novel, selective targeting toward the tumorigenic cells that survive to the anticancer agents. METHODS: Here, we have taken advantage of the therapeutic properties of an idiotype specific peptide (pA20-36) that bind specifically to murine B-lymphoma cells in the setting of an anti cancer strategy, based on the selected delivery of electrostatic-based complex, peptide-siRNA. To this end, two engineered, arginine rich, peptides that included the pA20-36 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide presented 9 Arg at the C-terminal of pA20-36 whereas the other included 5 Arg at the N- and C-terminus, respectively. RESULTS: Compared to the control and random peptide-siRNA complexes, both pA20-36-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry and confocal microscopy, whereas fluorescent-labelled siRNA alone was not internalized in the selected cells. Compared to peptide controls, the use of the modified pA20-36 peptides complexed with siRNA anti-GAPDH and anti-Bcl2 showed a down-regulation in the expression levels of the corresponding genes. CONCLUSIONS: Peptide-siRNA complex can be suitable tool for both selective peptide-driven cell targeting and gene silencing. In this setting, the improvement of this strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.

Aberrant glycosylation as biomarker for cancer: focus on CD43.

Glycosylation is a posttranslational modification of proteins playing a major role in cell signalling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure variability and binding specificity to lectin ligands. Aberrant expression of glycan structures as well as occurrence of truncated structures, precursors, or novel structures of glycan may affect ligand-receptor interactions and thus interfere with regulation of cell adhesion, migration, and proliferation. Indeed, aberrant glycosylation represents a hallmark of cancer, reflecting cancer-specific changes in glycan biosynthesis pathways such as the altered expression of glycosyltransferases and glycosidases. Most studies have been carried out to identify changes in serum glycan structures. In most cancers, fucosylation and sialylation are significantly modified. Thus, aberrations in glycan structures can be used as targets to improve existing serum cancer biomarkers. The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. In this review, we discuss the aberrant protein glycosylation associated with human cancer and the identification of protein glycoforms as cancer biomarkers. In particular, we will focus on the aberrant CD43 glycosylation as cancer biomarker and the potential to exploit the UN1 monoclonal antibody (UN1 mAb) to identify aberrant CD43 glycoforms.

FKBP51 increases the tumour-promoter potential of TGF-beta.

BACKGROUND: FKBP51 (FKBP5 Official Symbol) is a large molecular weight component of the family of FK506 binding proteins (FKBP). In recent years, research studies from our laboratory highlighted functions for FKBP51 in the control of apoptosis and melanoma progression. FKBP51 expression correlated with the invasiveness and aggressiveness of melanoma. Since a role for TGF-ß in the enhanced tumorigenic potential of melanoma cells is widely described, we hypothesized a cooperative effect between FKBP51 and TGF-ß in melanoma progression. METHODS: SAN and A375 melanoma cell lines were utilized for this study. Balb/c IL2γ NOD SCID served to assess the ability to colonize organs and metastasize of different cell lines, which was evaluated by in vivo imaging. Realtime PCR and western blot served for measurement of mRNA and protein expression, respectively. RESULTS: By comparing the metastatic potential of two melanoma cell lines, namely A375 and SAN, we confirmed that an increased capability to colonize murine organs was associated with increased levels of FKBP51. A375 melanoma cell line expressed FKBP51 mRNA levels 30-fold higher in comparison to the SAN mRNA level and appeared more aggressive than SAN melanoma cell line in an experimental metastasis model. In addition, A375 expressed, more abundantly than SAN, the TGF-ß and the pro angiogenic TGF-ß receptor type III (TßRIII) factors. FKBP51 silencing produced a reduction of TGF-ß and TßRIII gene expression in A375 cell line, in accordance with previous studies. We found that the inducing effect of TGF-ß on Sparc and Vimentin expression was impaired in condition of FKBP51 depletion, suggesting that FKBP51 is an important cofactor in the TGF-ß signal. Such a hypothesis was supported by co immunoprecipitation assays, showing that FKBP51 interacted with either Smad2,3 and p300. In normal melanocytes, FKBP51 potentiated the effect of TGF-ß on N-cadherin expression and conferred a mesenchymal-like morphology to such round-shaped cells. CONCLUSIONS: Overall, our findings show that FKBP51 enhances some pro oncogenic functions of TGF-ß, suggesting that FKBP51-overexpression may help melanoma to take advantage of the tumor promoting activities of the cytokine.

Cancer-associated CD43 glycoforms as target of immunotherapy.

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy.

Validation of microarray data in human lymphoblasts shows a role of the ubiquitin-proteasome system and NF-kB in the pathogenesis of Down syndrome.

BACKGROUND: Down syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown. METHODS: To better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade. RESULTS: We show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes. CONCLUSIONS: The present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS.

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB.

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.

Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome.

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) - a natural polyphenol component of green tea - to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content. In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome.

In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented.

Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome.

Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.

Analysis of seven maternal polymorphisms of genes involved in homocysteine/folate metabolism and risk of Down syndrome offspring.

PURPOSE: We present a case-control study of seven polymorphisms of six genes involved in homocysteine/folate pathway as risk factors for Down syndrome. Gene-gene/allele-allele interactions, haplotype analysis and the association with age at conception were also evaluated. METHODS: We investigated 94 Down syndrome-mothers and 264 control-women from Campania, Italy. RESULTS: Increased risk of Down syndrome was associated with the methylenetetrahydrofolate reductase (MTHFR) 1298C allele (OR 1.46; 95% CI 1.02-2.10), the MTHFR 1298CC genotype (OR 2.29; 95% CI 1.06-4.96), the reduced-folate-carrier1 (RFC1) 80G allele (1.48; 95% CI 1.05-2.10) and the RFC1 80 GG genotype (OR 2.05; 95% CI 1.03-4.07). Significant associations were found between maternal age at conception > or = 34 years and either the MTHFR 1298C or the RFC 180G alleles. Positive interactions were found for the following genotype-pairs: MTHFR 677TT and 1298CC/CA, 1298CC/CA and RFC1 80 GG/GA, RFC1 80 GG and methylenetetrahydrofolate-dehydrogenase 1958 AA. The 677-1298 T-C haplotype at the MTHFR locus was also a risk factor for Down syndrome (P = 0.0022). The methionine-synthase-reductase A66G, the methionine-synthase A2756G and the cystathionine-beta-synthase 844ins68 polymorphisms were not associated with increased risk of Down syndrome. CONCLUSION: These results point to a role of maternal polymorphisms of homocysteine/folate pathway as risk factors for Down syndrome.