Alessandra Cirillo

The Expression of Caspases is Enhanced in Peripheral Blood Mononuclear Cells of Autism Spectrum Disorder Patients

Autism and autism spectrum disorders (ASDs) are heterogeneous complex neuro-developmental disorders characterized by dysfunctions in social interaction and communication skills. Their pathogenesis has been linked to interactions between genes and environmental factors. Consistent with the evidence of certain similarities between immune cells and neurons, autistic children also show an altered immune response of peripheral blood mononuclear cells (PBMCs). In this study, we investigated the activation of caspases, cysteinyl aspartate-specific proteases involved in apoptosis and several other cell functions in PBMCs from 15 ASD children compared to age-matched normal healthy developing controls. The mRNA levels for caspase-1, -2, -4, -5 were significantly increased in ASD children as compared to healthy subjects. Protein levels of Caspase-3, -7, -12 were also increased in ASD patients. Our data are suggestive of a possible role of the capsase pathway in ASD clinical outcome and of the use of caspase as potential diagnostic and/or therapeutic tools in ASD management.

Epigenetic Findings in Autism: New Perspectives for Therapy

Autism and autism spectrum disorders (ASDs) are complex neurodevelopmental disorders characterized by dysfunctions in social interactions, communications, restricted interests, and repetitive stereotypic behaviors. Despite extensive genetic and biological research, significant controversy surrounds our understanding of the specific mechanisms of their pathogenesis. However, accumulating evidence points to the involvement of epigenetic modifications as foundational in creating ASD pathophysiology. Epigenetic modifications or the alteration of DNA transcription via variations in DNA methylation and histone modifications but without alterations in the DNA sequence, affect gene regulation. These alterations in gene expression, obtained through DNA methylation and/or histone modifications, result from transcriptional regulatory influences of environmental factors, such as nutritional deficiencies, various toxicants, immunological effects, and pharmaceuticals. As such these effects are epigenetic regulators which determine the final biochemistry and physiology of the individual. In contrast to psychopharmacological interventions, bettering our understanding of how these gene-environmental interactions create autistic symptoms should facilitate the development of therapeutic targeting of gene expression for ASD biomedical care.

Autism Spectrum Disorders: Is Mesenchymal Stem Cell Personalized Therapy the Future?

Autism and autism spectrum disorders (ASDs) are heterogeneous neurodevelopmental disorders. They are enigmatic conditions that have their origins in the interaction of genes and environmental factors. ASDs are characterized by dysfunctions in social interaction and communication skills, in addition to repetitive and stereotypic verbal and nonverbal behaviours. Immune dysfunction has been confirmed with autistic children. There are no defined mechanisms of pathogenesis or curative therapy presently available. Indeed, ASDs are still untreatable. Available treatments for autism can be divided into behavioural, nutritional, and medical approaches, although no defined standard approach exists. Nowadays, stem cell therapy represents the great promise for the future of molecular medicine. Among the stem cell population, mesenchymal stem cells (MSCs) show probably best potential good results in medical research. Due to the particular immune and neural dysregulation observed in ASDs, mesenchymal stem cell transplantation could offer a unique tool to provide better resolution for this disease.

Comparison of X‐chromosome inactivation in Duchenne muscle/myocardium‐manifesting carriers, non‐manifesting carriers and related daughters

Female carriers of Duchenne muscular dystrophy (DMD) are usually asymptomatic. However, 2.5–7.8% of them may present muscle symptoms and cardiomyopathy, attributed to a reduced production of dystrophin, probably because of skewed patterns of X‐chromosome inactivation (XCI). To evaluate the role of XCI in symptomatic (at muscle or heart level) and asymptomatic DMD carriers, 44 subjects were selected from our database (12 manifesting, 21 non‐manifesting, 11 healthy females), and XCI pattern determined in the lymphocytes by the androgen receptor methylation‐based assay. The results showed that DMD‐manifesting carriers had a preferential inactivation of the X‐chromosome carrying the normal allele, while non‐manifesting carriers and healthy females showed a random XCI pattern. Moreover, when comparing muscle with heart manifesting carriers, the former group showed a higher degree of skewing. No concordance in XCI was found between mothers and daughters, when symptomatic/asymptomatic mother–daughter pairs were analyzed. The results confirm that DMD clinical manifestations in carriers are associated with non‐random patterns of X inactivation.

The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages

BackgroundImmune system dysregulation is well-recognized in autism and thought to be part of the etiology of this disorder. The endocannabinoid system is a key regulator of the immune system via the cannabinoid receptor type 2 (CB2R) which is highly expressed on macrophages and microglial cells. We have previously published significant differences in peripheral blood mononuclear cell CB2R gene expression in the autism population. The use of the Gc protein-derived Macrophage Activating Factor (GcMAF), an endogenous glycosylated vitamin D binding protein responsible for macrophage cell activation has demonstrated positive effects in the treatment of autistic children. In this current study, we investigated the in vitro effects of GcMAF treatment on the endocannabinoid system gene expression, as well as cellular activation in blood monocyte-derived macrophages (BMDMs) from autistic patients compared to age-matched healthy developing controls.MethodsTo achieve these goals, we used biomolecular, biochemical and immunocytochemical methods.ResultsGcMAF treatment was able to normalize the observed differences in dysregulated gene expression of the endocannabinoid system of the autism group. GcMAF also down-regulated the over-activation of BMDMs from autistic children.ConclusionsThis study presents the first observations of GcMAF effects on the transcriptionomics of the endocannabinoid system and expression of CB2R protein. These data point to a potential nexus between endocannabinoids, vitamin D and its transporter proteins, and the immune dysregulations observed with autism.

High grade glioblastoma is associated with aberrant expression of ZFP57, a protein involved in gene imprinting, and of CPT1A and CPT1C that regulate fatty acid metabolism.

The diagnosis of glioblastoma is still based on tumor histology, but emerging molecular diagnosis is becoming an important part of glioblastoma classification. Besides the well-known cell cycle-related circuitries that are associated with glioblastoma onset and development, new insights may be derived by looking at pathways involved in regulation of epigenetic phenomena and cellular metabolism, which may both be highly deregulated in cancer cells. We evaluated if in glioblastoma patients the high grade of malignancy could be associated with aberrant expression of some genes involved in regulation of epigenetic phenomena and lipid metabolism. We measured the mRNA levels of ZFP57, TRIM28, CPT1A, CPT1B, and CPT1C in a cohort of 80 patients divided in two groups: grade II and grade IV. We evidenced that high grade glioblastoma is associated with increased level of ZFP57, a protein involved in gene imprinting, and aberrant expression of CPT1A and CPT1C, regulators of fatty acid oxidation. Our study may pave the way to identify new markers that could be potentially useful for diagnosis and/or prognosis of glioblastoma.

A preamplification approach to GMO detection in processed foods

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.

Beneficial Effects of Palmitoylethanolamide on Expressive Language, Cognition, and Behaviors in Autism: A Report of Two Cases.

Introduction. Autism spectrum disorders are defined by behavioral and language atypias. Growing body of evidence indicates inflammatory mediators may contribute to the condition. Palmitoylethanolamide (PEA) is naturally occurring and has been available as a nonprescription medical food supplement in Europe since 2008. PEA has been tested in thousands of human subjects without any noted significant side effects. Here we report the first cases of the administration of PEA to two children with autism. Case Presentations. The first 13-year-old male child (Subject 1) presented with a total IgE of 572 IU/mL (nl < 200) and with low mature CD57+ natural killer cell counts (32 cells/µL; nl = 60–300 cells/µL) and with significant eczema and allergic stigmata. Expressive language, as measured by mean length of utterance, and overall autism severity as measured by the Childhood Autism Rating Scale, Second Edition, improved significantly. Atopic symptoms diminished. No side effects were reported. The second male child, age 15 (Subject 2), also displayed noticeable and rapid improvements in cognitive, behaviors, and sociability. Conclusion. Currently, there is no definitive treatment for autism condition. Palmitoylethanolamide could be an effective treatment for autism syndrome. We propose appropriate double-blind clinical trials to further explore palmitoylethanolamide efficacy and safety.

Preamplification procedure for the analysis of ancient DNA samples.

In ancient DNA studies the low amount of endogenous DNA represents a limiting factor that often hampers the result achievement. In this study we extracted the DNA from nine human skeletal remains of different ages found in the Byzantine cemetery of Abdera Halkidiki and in the medieval cemetery of St. Spiridion in Rhodes (Greece). Real-time quantitative polymerase chain reaction (qPCR) was used to detect in the extracts the presence of PCR inhibitors and to estimate the DNA content. As mitochondrial DNA was detected in all samples, amplification of nuclear targets, as amelogenin and the polymorphism M470V of the transmembrane conductance regulator gene, yielded positive results in one case only. In an effort to improve amplification success, we applied, for the first time in ancient DNA, a preamplification strategy based on TaqMan PreAmp Master Mix. A comparison between results obtained from nonpreamplified and preamplified samples is reported. Our data, even if preliminary, show that the TaqMan PreAmp procedure may improve the sensitivity of qPCR analysis.

Skewed X-chromosome inactivation plays a crucial role in the onset of symptoms in carriers of Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is an X‐linked recessive disorder affecting approximately 1: 18.000 male births. Female carriers are usually asymptomatic, although 2.5–18% may present muscle or heart symptoms. In the present study, the role of the X chromosome inactivation (XCI) on the onset of symptoms in BMD carriers was analysed and compared with the pattern observed in Duchenne muscular dystrophy (DMD) carriers.