Congenital heart defects (CHD) have an incidence of 5–8/1000 live births1, are six times more common than chromosomal abnormalities and are four times more common than neural tube defects2. Approximately half of cases of CHD are severe, usually requiring one or more surgical procedures in the neonatal period or during childhood3. Fetal echocardiography during the first or second trimester is considered the best screening tool for CHD, with a pooled sensitivity and specificity of 85% (95% CI, 78–90%) and 99% (95% CI, 98–100%), respectively, as reviewed by Rasiah et al.4. However, at present, the use of ultrasound examination for the detection of major CHD is limited to a few specialized and experienced centers, where it may be offered to high-risk mothers5. For this reason, it is more common to screen for fetal CHD using various biomarkers, such as nuchal translucency (NT), beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A (PAPP-A), but these are burdened by a high rate of false-positive results. A recent paper by Llurba et al.6 introduced the possibility of using as a new marker to screen for CHD circulating placental growth factor, found in maternal blood. We are glad that the paper refers to our hypothesis about the correlation between some circulating messenger RNA (mRNA) species and CHD7. There is very little relevant information in the literature; however, we wish to highlight two papers that could support the fact that the genetic profile from chorionic villus sampling (CVS) reflects that of intraembryonic mesoderm derivates, such as the heart and the NT. In the first paper8, we explored the genetic profile obtained from CVS in four fetuses with an enlarged NT (> 95th centile) but normal karyotype. Interestingly, abnormal NT cases displayed a genetic CVS profile consistent with the proposed etiopathogenesis of abnormal NT9. In particular, the presence of aberrant amounts of mRNA for some genes related to extracellular matrix structure and metabolism (tissue inhibitor of metalloproteinase 1 (TIMP1), disintegrin-like and metalloprotease reprolysin type with thrombospondin type 1 motif, 2 (ADAMTS2)) might be responsible for transitory abnormal connective tissue development. Again, the underexpression of genes such as endothelin 3 (EDN3) supports the supposed poor migration of neural cells that may occur in association with abnormal NT10. Briefly, given such results, it seems reasonable that extraembryonic mesoderm (from which the mesenchymal axis of the villus develops) and intraembryonic mesoderm (from which the NT arises) have quite similar genetic expression during the first trimester of human fetal development. The next step at the basis of this hypothesis was to demonstrate that the fetal heart (another intraembryonic mesoderm derivate) and the placenta could also share a similar profile. In our study7, we selected some fetuses affected by CHDs (mainly interventricular septal defects), and analysis of placentae collected after delivery revealed an aberrant genetic profile. The abnormal profiles detected in these placentae corresponded to certain genes involved in cardiac morphogenesis and congestive heart failure. Finally, and more importantly, in a new series of women bearing a fetus affected with a CHD, some of the mRNA species previously found to have aberrant levels of gene expression in placental tissues in fact showed a different concentration in the circulating maternal blood, and this concentration was found to be statistically different when compared with that in controls. For some of them, such as salvador homolog 1 protein (SAV1), tenascin (TNXB), PAPP-A and β-myosin heavy chain (MYL7), it was also possible to calculate a detection rate for a fixed 10% false-positive rate. This idea opens up new possibilities in the field of CHD screening, as reported in a recent review by Yu et al.11. A major question mark remains regarding the source of some of these circulating mRNA species. In fact, if the extraembryonic and intraembryonic mesoderm have a similar origin, not only the trophoblast but also the mesenchymal villous axis should contribute to the pool of circulating fetal nucleic acids in maternal blood. This hypothesis could also have an impact on the prenatal diagnosis of fetal aneuploidies by fetal DNA dosage in which, in substance, only the trophoblast is conventionally considered a source of fetal genetic material. To conclude, mRNA species possibly originating from both the trophoblast and the mesenchymal axis can be used to screen for CHD, probably in combination with biophysical and biochemical markers. A. Farina* and D. Arcelli Division of Prenatal Medicine, DIMEC University of Bologna, Bologna, Italy *Correspondence. (e-mail: [email protected])

Summary Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a rare syndrome due to a mutation in the forkhead box protein 3 gene (FOXP3) leading to an impaired regulatory T cell (Treg) activity associated both with skewed T helper type 2 (Th2) response and autoreactive phenomena. The purpose of this study was to describe a combined proteomics and genomics approach to comprehensively evaluate clinical and immunological phenotypes of patients affected by IPEX. T cell receptor (TCR)-Vβ repertoire and peripheral blood lymphocytes phenotype from three brothers affected by IPEX were studied by flow cytometry. Specific immunoglobulin (Ig)E were evaluated by means of an allergenic molecules microarray [immuno solid-phase allergen chip (ISAC)]. Total RNA was extracted and hybridized to Affymetrix oligonucleotide arrays to obtain quantitative gene-expression levels. No FOXP3 protein was detectable within CD127-CD25highCD4+ T cells from peripheral blood. A T cell-naive phenotype (CD62L+CD45R0-) associated with a reduction of both CD26 and CD7 expression and a TCR-Vβ 8 and 22 family expansions were found. B lymphocytes were mainly CD5+ (B1) cells expressing a naive phenotype (tcl1+CD27-). The three IPEX patients had severe food allergy and specific IgE reactivity to cow's milk allergens, a hen's egg allergen and a wheat allergen. Gene expression profile analysis revealed a dysregulation associated mainly with Th1/Th2 pathways. The multiplexing evaluation reported in this study represents a comprehensive approach in the assessment of genetic conditions affecting the immune system such as the IPEX syndrome, paving the way for the development of diagnostic tools to improve the standard clinical and immunological profiling of the disease.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma and its pathogenesis is still unknown. Diagnosis/prognosis may strongly ameliorate the management of SS individuals. Here, we profiled the expression of 470 microRNAs (miRNAs) in a cohort of 22 SS patients, and we identified 45 miRNAs differentially expressed between SS and controls. Using predictive analysis, a list of 19 miRNAs, including miR-21, miR-214, miR-486, miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs including again miR-21, potentially able to discriminate patients with unfavorable and favorable outcome. We validated our data for miR-21, miR-214 and miR-486 by qRT-PCR, including an additional set of array-independent SS cases. In addition, we also provide an in vitro evidence for a contribution of miR-214, miR-486 and miR-21 to apoptotic resistance of CTCL cell line.

To calculate the risk of developing pre‐eclampsia (PET) in a consecutive series of low‐risk women at 18–24 weeks' gestation, using recently published logistic regression models.

BACKGROUND:Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM:To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS:Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS:A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS:This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.

Objective To retrospectively investigate whether the genetic profile from chorionic villous sampling (CVS) found in euploid fetuses with increased NT differs from matched controls.Study Design We employed cDNA microarray technology to characterize and compare the gene expression profile of chorionic villous tissues (which encompass the trophoblast and inner mesenchymal core) belonging to four singleton male fetuses with increased NT at 10-11 weeks' gestation. A pool of four normal chorionic villous tissues belonging to four respective fetuses, matched for gestational age and gender, was used as controls.Results In euploid fetuses, we found several underexpressed genes, possibly involved in mechanisms associated with the abnormal NT thickness. All these genes are likely to belong to the mesenchymal core of the villus that originates from the extraembryonic mesoderm, and thus might be closely representative of the embryonic genetic profile. They include: (1) genes of embryonic development and differentiation such as Endothelin 3 (EDN3) and secreted frizzled-related protein 4 (SFRP4); (2) genes of the extracellular matrix (ECM) metabolism such as tissue inhibitor of metalloproteinase 1 (TIMP1), and disintegrin-like and matrix metalloproteinase (MMP) (reprolysin type) with thrombospondin type 1 Motif or ADAMTS2, exostoses (multiple)-like 1 (EXTI-1), heparan sulfate (HS) 6-O-sulfotransferase 1 or HS6ST1, fibronectin 1 (FN1) and Integrin Alpha 10 (ITGA10) involved in HS and proteoglycan bio-synthesis, ECM synthesis and cell-matrix adhesion; (3) genes involved in vessel formation and differentiation such as angiogenic factor (VG5Q), and in blood pressure control and muscle contraction, like Endothelin 3 or EDN3 and sarcolemma associated protein (SLMAP). Such lower expressions of the villous tissues might be related to an immature genetic profile of the embryo development as well as abnormal regulation of ECM bio-synthesis and/or improper vessel growth and blood pressure control. Also, the results partially support the theories proposed for NT enlargement such as altered composition of ECM and abnormal/delayed development of the circulatory system.Conclusions Abnormal extraembryonic genetic expression is found at 10-11 weeks' gestation in euploid fetuses with increased NT. If both extra- and intraembryonic mesoderms express the same genetic alterations, then microarray analyses on CVS could be used to screen several mesoderm-derivate anomalies. (c) Copyright 2006 John Wiley & Sons, Ltd.

Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma (CTCL) characterized by a distinct metastatic pattern mainly involving blood and skin. Chemokines and their receptors play a critical role in cellular recruitment and homing to tissues and in the metastatic process of several tumors including non-Hodgkin T-cell lymphomas (NHLs). Here we report that SS cells express a functionally active CXCR4 and that its ligand SDF-1 is abundantly produced in the skin, which represents the main destination of SS cell spreading. SDF-1 is normally inactivated by proteolytic cleavage by the CD26/dipeptidylpeptidase IV (DPPIV). The lack of CD26 from the cell surface is a hallmark of circulating SS cells. We also show that the CD26(-) phenotype is maintained also in skin-infiltrating neoplastic T lymphocytes and that SS-affected individuals exhibit a reduced activity of plasma soluble CD26. Finally, we observe that the addition of soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26(+) CTCL cell line, enhances the SDF-1-induced migration of these cells. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.

Sezary Syndrome (SS) is a rare form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving skin and blood. Chemokine and chemokine receptors have been implicated in the spreading process of many cancers including various forms of non-Hodgkin T-cell lymphomas (NHL). In this study we report that chemokine receptor CXCR4 is over-expressed by both circulating and skin-homing neoplastic T-lymphocytes of SS patients and is functionally active as demonstrated by the migration of freshly isolated Sezary (SzS) cells along the chemical gradient of its natural ligand SDF-1. To shed light on the regulation of CXCR4/SDF1 interaction, we also investigated the enzymatic activity of CD26/dipeptidylpeptidase IV (DPPIV) since SDF-1 is efficiently inactivated by CD26, in physiological condition.. This is of particular relevance because one of the hallmark of the circulating SzS cells is the loss of CD26 from the cell surface. We first demonstrated that the CD26 negative phenotype is similarly maintained also in the skin-homing neoplastic T lymphocytes; we then observed that the addition of exogenus soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26-positive CTCL cell line, enhances the SDF-1-induced migration of these cells. We finally showed that SS individuals exhibit a reduced activity of the soluble CD26 as revealed by the measurements performed on the patients derived plasma. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.

Background: P‐15 is an analog of the cell‐binding domain of collagen. P‐15 has been shown to facilitate physiological processes in a way similar to collagen; to serve as an anchorage for cells; and to promote the binding, migration, and differentiation of cells. Methods: Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast‐like cell line (MG‐63) cultured with P‐15 several genes whose expression was significantly up‐ or downregulated. Results: The differentially expressed genes cover a broad range of functional activities: 1) signaling transduction, 2) differentiation, 3) apoptosis, and 4) cell‐cycle regulation. It was also possible to detect some genes whose function is unknown. Conclusions: The data reported are, to our knowledge, the first genetic portrait of P‐15 effects. They can help us to better understand the molecular mechanism of osteogenesis and can serve as a model for comparing different cell cultures and/or other materials with similar effect. J Periodontol 2004;75:66‐83 .

Cisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti. To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase. As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels. These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.

One of the most common problems encountered while deciphering results from expression profiling experiments is in relating differential expression of genes to molecular functions and cellular processes. A second important problem is that of comparing experiments performed by different labs using different microarray platforms, or even unrelated techniques. Gene Ontology (GO) is now used to describe biological features, since GO terms are associated with genes, to overcome the apparent distance between expression profiles and biological comprehension. Here we describe the development, implementation and use of GOAL (Gene Ontology Automated Lexicon), a web-based application for the identification of functions and processes regulated in microarray and SAGE (serial analysis of gene expression) experiments. We applied GOAL to a range of experimental datasets related to different biological problems, including cancer and the cell cycle. By using GOAL, reported and novel relevant processes were identified in a number of experiments by our collaborators and by us. Different datasets could also be compared with each other to define conserved functional modules. GOAL allows a seamless and high-level analysis of expression profiles and is implemented as a free WWW resource (http://microarrays.unife.it).

Surface implant modifications have been shown to have a relevant importance in modifying cell response. Expression profiling by DNA microarray is a new molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) on new implant surface (nanoPORE, Out-Link, Sweden and Martina, Due Carrare, Padova, Italy), several genes whose expressions were significantly down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport (c) apoptosis and cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of an implant surface. They can be relevant to better understand the molecular mechanism of implant osseointegration and as a model for comparing other materials.

Odontogenic tumors are rare neoplasms arising from the odontogenic apparatus. We aimed to identify molecular characteristics associated with odontogenic tumorigenesis and malignancy. To this end, we investigated the expression level of human genes by using, for the first time in odontogenic tumors, the technique of expression profiling. Gene expression alterations common to all six odontogenic tumors were identified by the use of cDNA microarrays containing 19,000 human cDNAs. Statistical analysis on a subset of 4974 cDNAs present in the biopsies identified 506 distinct genes associated with the tumors (p-value < 0.01). Gene ontology analysis of the cellular processes which were differentially regulated in odontogenic tumors was accomplished by the use of a subset of 1409 annotated genes. Finally, 43 cDNAs differentiated the three malignant odontogenic tumors (ameloblastic carcinoma, clear cell odontogenic tumor, granular cell odontogenic tumor) from the three benign ameloblastoma biopsies (p < 0.01). The identified genes might help us better classify borderline odontogenic tumors.

In the head and neck region, clear cell tumors are usually derived from salivary glands, odontogenic tissues, and metastasis. The World Health Organization has classified clear cell odontogenic tumor among benign tumors, but it is now recognized as a more sinister lesion, and current opinion is that it should be designated as a carcinoma. It is characterized by aggressive growth, recurrences, and metastasis. By using complementary DNA microarrays, several genes in clear cell odontogenic tumor were identified that are differentially regulated when compared with non-tumor tissue. In conclusion, the first genetic profiling of clear odontogenic carcinoma is reported. DNA microarrays can potentially help in identifying some genes whose products could be disease-specific targets for cancer therapy as well as a tool for better classifying odontogenic tumor.

Zirconium oxide ceramics have outstanding mechanical properties, a high biocompatibility and a high resistance to scratching. Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured on zirconium oxide discs (Cercon, Degussa Dental, Hanau, Germany) several genes whose expression was significantly up or down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport and (c) cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of a zirconium oxide surface. They can be relevant to better understand the molecular mechanism of biocompatibility and as a model for comparing other materials.

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