Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

UNLABELLED: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. SIGNIFICANCE: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.

Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models.

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca(2+) increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca(2+) increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role.

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

Clinical pharmacogenetics of methotrexate.

It is well known that interindividual variability can affect the response to many drugs in relation to age, gender, diet, and organ function. Pharmacogenomic studies have also documented that genetic polymorphisms can exert clinically significant effects in terms of drug resistance, efficacy and toxicity by modifying the expression of critical gene products (drug-metabolizing enzymes, transporters, and target molecules) as well as pharmacokinetic and pharmacodynamic parameters. A growing body of in vitro and clinical evidence suggests that common polymorphisms in the folate gene pathway are associated with an altered response to methotrexate (MTX) in patients with malignancy and autoimmune disease. Such polymorphisms may also induce significant MTX toxicity requiring expensive monitoring and treatment. Although the available data are not conclusive, they suggest that in the future MTX pharmacogenetics could play a key role in clinical practice by improving and tailoring treatment. This review describes the genetic polymorphisms that significantly influence MTX resistance, efficacy, and toxicity.

Differential screening of phage-ab libraries by oligonucleotide microarray technology.

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.