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Background: Patients with nonischemic dilated cardiomyopathy (DCM), severe left ventricular (LV) dysfunction, and complete left bundle branch block benefit from cardiac resynchronization therapy (CRT). However, a large heterogeneity of response to CRT is described. Several predictors of response to CRT have been identified, but the role of the underlying genetic background is still poorly explored., Objectives: In the present study, the authors sought to define differences in LV remodeling and outcome prediction after CRT when stratifying patients according to the presence or absence of DCM-causing genetic background., Methods: From our center, 74 patients with DCM subjected to CRT and available genetic testing were retrospectively enrolled. Carriers of causative monogenic variants in validated DCM-causing genes, and/or with documented family history of DCM, were classified as affected by genetically determined disease (GEN+DCM) (n = 25). Alternatively, by idiopathic dilated cardiomyopathy (idDCM) (n = 49). The primary outcome was long-term LV remodeling and prevalence of super response to CRT (evaluated at 24-48 months after CRT); the secondary outcome was heart failure-related death/heart transplant/LV assist device., Results: GEN+DCM and idDCM patients were homogeneous at baseline with the exception of QRS duration, longer in idDCM. The median follow-up was 55 months. Long-term LV reverse remodeling and the prevalence of super response were significantly higher in the idDCM group (27% in idDCM vs 5% in GEN+DCM; P = 0.025). The heart failure-related death/heart transplant/LV assist device outcome occurred more frequently in patients with GEN+DCM (53% vs 24% in idDCM; P = 0.028)., Conclusions: Genotyping contributes to the risk stratification of patients with DCM undergoing CRT implantation in terms of LV remodeling and outcomes., Competing Interests: Funding Support and Author Disclosures This study was supported by CRTrieste Foundation and Cassa di Risparmio di Gorizia Foundation to Dr Sinagra. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Erythropoietic protoporphyria is a rare disorder which represents an important health problem in children, causing painful photosensitivity. Little is known on the correlation between genetic profile and clinical manifestations. The standard of care for Erythropoietic protoporphyria is based on avoiding sun and using sun protections, but recent literature has suggested that cimetidine may have a role in improving sun sensitivity. Herein we report our case series describing the successful use of cimetidine and analyzing potential phenotype-genotype correlations., Case Presentation: This case series describes five patients presented to our Rheumatology Service complaining sun sensitivity. Blood exams and genetic analysis were consistent with the diagnosis of erythropoietic protoporphyria. Four of 5 patients received cimetidine in addition to standard therapies and the effect of treatment was evaluated by Erythropoietic Protoporphyria - Quality of Life questionnaire., Conclusions: Erythropoietic protoporphyria usually manifests in early childhood after a short sun exposure. Skin manifestations are the main reason for investigations, although sometimes they can be more subtle, leading to a significant diagnostic delay. Skin diseases in children can have profound effects on their family and social relationships. A treatment with cimetidine appears to be an excellent therapeutic option in children with Erythropoietic protoporphyria., (© 2023. The Author(s).)

Inborn errors of immunity (IEI) associated with immune dysregulation are not sufficiently addressed in shared recommendation, resulting in delayed diagnosis and high morbidity. The availability of precision medicine for some of these immune defects makes it urgent to evaluate effective strategies to diagnose and treat such defects before the occurrence of severe complications. A diagnosis of an IEI in these patients enabled the use of a more specific treatment in most cases, and these have the potential to prevent further disease progression. We studied immune dysregulation diseases in 30 patients with autoimmune or allergic phenotypes, exploiting data from clinics and immunophenotype, genetic and transcriptome investigations, and 6 of them were diagnosed with a monogenic disorder. Our results confirm that a non-negligible number of children with IEIs may present with signs and symptoms of immune dysregulation and share many features with common multifactorial immune conditions. Reaching a genetic diagnosis becomes more likely in the presence of multiple clinical manifestations, especially when in association with abnormalities of lymphocytes subsets and/or immunoglobulins levels. Moreover, 5 of 6 patients that obtained a diagnosis of monogenic disorder received precision therapy, in four cases with a good or moderate response.

Background: Diverse genetic backgrounds often lead to phenotypic heterogeneity in cardiomyopathies (CMPs). Previous genotype-phenotype studies have primarily focused on the analysis of a single phenotype, and the diagnostic and prognostic features of the CMP genotype across different phenotypic expressions remain poorly understood., Objectives: We sought to define differences in outcome prediction when stratifying patients based on phenotype at presentation compared with genotype in a large cohort of patients with CMPs and positive genetic testing., Methods: Dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy, left-dominant arrhythmogenic cardiomyopathy, and biventricular arrhythmogenic cardiomyopathy were examined in this study. A total of 281 patients (80% DCM) with pathogenic or likely pathogenic variants were included. The primary and secondary outcomes were: 1) all-cause mortality (D)/heart transplant (HT); 2) sudden cardiac death/major ventricular arrhythmias (SCD/MVA); and 3) heart failure-related death (DHF)/HT/left ventricular assist device implantation (LVAD)., Results: Survival analysis revealed that SCD/MVA events occurred more frequently in patients without a DCM phenotype and in carriers of DSP, PKP2, LMNA, and FLNC variants. However, after adjustment for age and sex, genotype-based classification, but not phenotype-based classification, was predictive of SCD/MVA. LMNA showed the worst trends in terms of D/HT and DHF/HT/LVAD., Conclusions: Genotypes were associated with significant phenotypic heterogeneity in genetic cardiomyopathies. Nevertheless, in our study, genotypic-based classification showed higher precision in predicting the outcome of patients with CMP than phenotype-based classification. These findings add to our current understanding of inherited CMPs and contribute to the risk stratification of patients with positive genetic testing., Competing Interests: Funding Support and Author Disclosures This study was supported by National Institutes of Health (NIH) grants R01HL69071, R01HL116906, and R01HL147064, and American Heart Association grant 17GRNT33670495 (to Dr Mestroni); NIH grant 1K23HI067915; NIH grants 2UM1HG006542 and R01HL109209 (to Dr Taylor); and CRTrieste Foundation and Cassa di Risparmio di Gorizia Foundation (to Dr Sinagra). This work is also supported by NIH/National Center for Advancing Translational Sciences Colorado CTSA grant numbers UL1 TR002535 and UL1 TR001082. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Recurrent aphthous stomatitis with systemic signs of inflammation can be encountered in inflammatory bowel disease, Behçet's disease (BD), Systemic Lupus Erythematosus (SLE). In addition, it has been proposed that cases with very early onset in childhood can be underpinned by rare monogenic defects of immunity, which may require targeted treatments. Thus, subjects with early onset recurrent aphthous stomatitis receiving a clinical diagnosis of BD-like or SLE-like disease may deserve a further diagnostic workout, including immunologic and genetic investigations., Objective: To investigate how an immunologic, genetic and transcriptomics assessment of interferon inflammation may improve diagnosis and care in children with recurrent aphthous stomatitis with systemic inflammation., Methods: Subjects referred to the pediatric rheumatologist for recurrent aphthous stomatitis associated with signs of systemic inflammation from January 2015 to January 2020 were enrolled in the study and underwent analysis of peripheral lymphocyte subsets, sequencing of a 17-genes panel and measure of interferon score., Results: We enrolled 15 subjects (12 females, median age at disease onset 4 years). The clinical diagnosis was BD in 8, incomplete BD in 5, BD/SLE overlap in 1, SLE in 1. Pathogenic genetic variants were detected in 3 patients, respectively 2 STAT1 gain of function variants in two patients classified as BD/SLE overlap and SLE, and 1 TNFAIP3 mutation (A20 haploinsufficiency) in patients with BD. Moreover 2 likely pathogenic variants were identified in DNASE1L3 and PTPN22, both in patients with incomplete BD. Interferon score was high in the two patients with STAT1 GOF mutations, in the patient with TNFAIP3 mutation, and in 3 genetic-negative subjects. In two patients, the treatment was modified based on genetic results., Conclusions: Although recurrent aphthous stomatitis associated with systemic inflammation may lead to a clinical diagnosis of BD or SLE, subjects with early disease onset in childhood deserve genetic investigation for rare monogenic disorders. A wider genetic panel may help disclosing the genetic background in the subset of children with increased interferon score, who tested negative in this study.

Primary immunodeficiencies (PIDs) are a large and growing group of disorders commonly associated with recurrent infections. However, nowadays, we know that PIDs often carry with them consequences related to organ or hematologic autoimmunity, autoinflammation, and lymphoproliferation in addition to simple susceptibility to pathogens. Alongside this conceptual development, there has been technical advancement, given by the new but already established diagnostic possibilities offered by new genetic testing (e.g., next-generation sequencing). Nevertheless, there is also the need to understand the large number of gene variants detected with these powerful methods. That means advancing beyond genetic results and resorting to the clinical phenotype and to immunological or alternative molecular tests that allow us to prove the causative role of a genetic variant of uncertain significance and/or better define the underlying pathophysiological mechanism. Furthermore, because of the rapid availability of results, laboratory immunoassays are still critical to diagnosing many PIDs, even in screening settings. Fundamental is the integration between different specialties and the development of multidisciplinary and flexible diagnostic workflows. This paper aims to tell these evolving aspects of immunodeficiencies, which are summarized in five key messages, through introducing and exemplifying five clinical cases, focusing on diseases that could benefit targeted therapy.

: Nonischemic-dilated cardiomyopathy (NIDCM) is an entity that gathers extremely heterogeneous diseases. This awareness, although leading to continuous improvement in survival, has increased the complexity of NIDCM patients' management. Even though the endorsed 'red-flags' approach helps clinicians in pursuing an accurate etiological definition in clinical practice, it is not clear when and how peripheral centers should interact with referral centers with specific expertise in challenging scenarios (e.g. postmyocarditis and genetically determined dilated cardiomyopathy) and with easier access to second-line diagnostic tools and therapies. This position paper will summarize each step in NIDCM management, highlighting the multiple interactions between peripheral and referral centers, from first-line diagnostic workup and therapy to advanced heart failure management and long-term follow-up.

Cardiomyopathies are primary myocardial disorders, genetically determined, with clinical onset between the third and the fifth decade of life. They represent the main causes of sudden cardiac death and heart failure in the youth. The more common myocardial diseases in clinical practice are dilated cardiomyopathy, arrhythmogenic cardiomyopathy and hypertrophic cardiomyopathy. Next generation sequencing techniques, recently available for genetics researches, together with the diffusion of advanced imaging techniques, permitted in the last years a deeper knowledge of these pathologies. Nevertheless, diagnosis, etiology and several aspects of patients' clinical management remain complex and controversial. This review paper aims to propose some operative flow-charts, derived from scientific evidences and the internal protocol of the Cardiothoracovascular Department of Trieste Hospital, Italian referral Center for cardiomyopathies and heart failure, with more than 30 years of experience in diagnosis and management of patients who suffer from primary myocardial disorders.

Background: Platinum resistance is a major challenge in the management of ovarian cancer. Even low levels of acquired resistance at the cellular level lead to impaired response to cisplatin. In ovarian cancer intraperitoneal therapy, nanoparticle formulation can improve the cisplatin's pharmacokinetics and safety profile., Purpose: This work aimed to investigate the chemo-sensitivity of ovarian cancer SKOV3 cells upon short-term (72h) single treatment of cisplatin and cisplatin-loaded biodegradable nanoparticles (Cis-NP). The aim was then to determine the therapeutic properties of Cis-NP in vivo using a SKOV3-luc cells' xenograft model in mice., Methods: Cell cytotoxicity was assessed after the exposure of the cell culture to cisplatin or Cis-NP. The effect of treatments on EMT and CSC-like phenotype was studied by analyzing a panel of markers by flow cytometry. Intracellular platinum concentration was determined by inductively coupled plasma mass spectrometry (ICS-MS), and gene expression was evaluated by RNAseq analysis. The efficacy of intraperitoneal chemotherapy was evaluated in a SKOV3-luc cells' xenograft model in mice, through a combination of bioluminescence imaging, histological, and immunohistochemical analyses., Results: We observed in vitro that short-term treatment of cisplatin has a critical role in determining the potential induction of chemoresistance, and a nanotechnology-based drug delivery system can modulate it. The RNAseq analysis underlines a protective effect of nanoparticle system according to their ability to down-regulate several genes involved in chemoresistance, cell proliferation, and apoptosis. The highest intracellular platinum concentration obtained with Cis-NP treatment significantly improved the efficacy. Consistent with in vitro results, we found that Cis-NP treatment in vivo can significantly reduce tumor burden and aggressiveness compared to the free drug., Conclusion: Nanoparticle-mediated cisplatin delivery may serve as an intracellular depot impacting the cisplatin pharmacodynamic performance at cellular levels. These features may contribute to improving the drawbacks of conventional intraperitoneal therapy, and therefore will require further investigations in vivo., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Bortot et al.)

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N‑glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose‑6‑P to mannose‑1‑P, which results in low concentration levels of guanosine 5'‑diphospho‑D‑mannose (GDP‑Man), a nucleotide‑activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP‑Man‑loaded poly (D,L‑lactide‑co‑glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG‑Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α‑mannosidase, β‑glucoronidase and β‑galactosidase in defective and normal fibroblasts. GDP‑Man (30 µg/ml GDP‑Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α‑mannosidase and β‑galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of β‑glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG‑Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two‑dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP‑Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.

Purpose of Review: This review aims to summarize the current knowledge on the genetic background of dilated cardiomyopathy (DCM), with particular attention to the genotype-phenotype correlations and the possible implications for clinical management., Recent Findings: Next generation sequencing (NGS) has led to the identification of an increasing number of genes and mutations responsible for DCM. This genetic variability is probably related to the extreme heterogeneity of disease manifestation. Important findings have associated mutations of Lamin A/C (LMNA) and Filamin C (FLNC) to poor prognosis and the propensity to cause an arrhythmic phenotype, respectively. However, a deeper understanding of the genotype-phenotype correlation is necessary, because it could have several implications for the clinical management of the patients. Furthermore, the correct interpretation of pathogenicity of mutations and the clinical impact of genetic testing in DCM patients still represent important fields to be implemented. A pathogenic gene mutation can be identified in almost 40% of DCM patients. The recent discoveries and future research in the field of genotype-phenotype correlation may lead to a more personalized management of the mutation carriers towards the application of precision medicine in DCM.

Despite significant advances in cancer diagnostics and treatment, ovarian cancers (OC) continue to kill more than 150,000 women every year worldwide. Due to the relatively asymptomatic nature and the advanced stage of the disease at the time of diagnosis, OC is the most lethal gynecologic malignancy. The current treatment for advanced OC relies on the synergistic effect of combining surgical cytoreduction and chemotherapy; however, beside the fact that chemotherapy resistance is a major challenge in OC management, new imaging strategies are needed to target microscopic lesions and improve both cytoreductive surgery and patient outcomes. In this context, nanostructured probes are emerging as a new class of medical tool that can simultaneously provide imaging contrast, target tumor cells, and carry a wide range of medicines resulting in better diagnosis and therapeutic precision. Herein we summarize several exemplary efforts in nanomedicine for addressing unmet clinical needs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aim: To describe the frequency and clinical characteristics of patients with undifferentiated periodic fever (UPF) and to investigate whether a clinical classification of UPF based on the PRINTO-Eurofever score can help predicting the response to treatment and the outcome at follow-up., Methods: Clinical and therapeutic information of patients with recurrent fever who presented at a single pediatric rheumatology center from January 2006 through April 2016 were retrospectively collected. Patients with a clinical suspicion of hereditary periodic fever (HPF) syndrome and patients with clinical picture of periodic fever, aphthae, pharingitis, adenitis (PFAPA) who were refractory to tonsillectomy underwent molecular analysis of five HPF-related genes: MEFV (NM&#95;000243.2), MVK (NM&#95;000431.3), TNFRSF1A (NM&#95;001065.3), NLRP3 (NM&#95;001079821.2), NLRP12 (NM&#95;001277126.1). All patients who had a negative genetic result were defined as UPF and further investigated. PRINTO-Eurofever score for clinical diagnosis of HPF was calculated in all cases., Results: Of the 221 patients evaluated for periodic fever, twelve subjects with a clinical picture of PFAPA who were refractory to tonsillectomy and 22 subjects with a clinical suspicion of HPF underwent genetic analysis. Twenty-three patients (10.4%) resulted negative and were classified as UPF. The median age at presentation of patients with UPF was 9.5 mo (IQR 4-24). Patients with UPF had a higher frequency of aphthae (52.2% vs 0%, P = 0.0026) and musculoskeletal pain (65.2% vs 18.2%, P = 0.0255) than patients with genetic confirmed HPF. Also, patients with UPF had a higher frequency of aphthous stomatitis (52.2% vs 10.7%, P < 0.0001), musculoskeletal pain (65.2% vs 8,0%, P < 0.0001), and abdominal pain (52.2% vs 4.8%, P < 0.0001) and a lower frequency of pharyngitis (56.6% vs 81.3%, P = 0.0127) compared with typical PFAPA in the same cohort. Twenty-one of 23 patients with UPF (91.3%) received steroids, being effective in 16; 13 (56.2%) were given colchicine, which was effective in 6. Symptoms resolution occurred in 2 patients with UPF at last follow-up. Classification according to the PRINTO-Eurofever score did not correlate with treatment response and prognosis., Conclusion: UPF is not a rare diagnosis among patients with periodic fever. Clinical presentation place UPF half way on a clinical spectrum between PFAPA and HPF. The PRINTO-Eurofever score is not useful to predict clinical outcome and treatment response in these patients., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest.

In this work, iron/silica/gold core-shell nanoparticles (Fe 3 O 4 @SiO 2 @Au NPs) characterized by magnetic and optical properties have been synthesized to obtain a promising theranostic platform. To improve their biocompatibility, the obtained multilayer nanoparticles have been entrapped in polymeric micelles, decorated with folic acid moieties, and tested in vivo for photoacoustic and magnetic resonance imaging detection of ovarian cancer.

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44‑amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standard Ion AmpliSeq library preparation and Ion Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification.

Background: Periodic fever syndromes (PFS) are an emerging group of autoinflammatory disorders. Clinical overlap exists and multiple genetic analyses may be needed to assist diagnosis. We evaluated the diagnostic value of a 5-gene sequencing panel (5GP) in patients with undiagnosed PFS., Methods: Simultaneous double strand Sanger sequencing of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12 genes was performed in 42 patients with unexplained PFS. Clinical features were correlated with genetic results., Results: None of 42 patients analyzed displayed a causative genotype. However, single or multiple genetic variants of uncertain significance were detected in 24 subjects. Only in 5 subjects a definite diagnosis was made by taking into account both genetic and clinical data (2 TRAPS syndrome; 2 FMF; 1 FCAS). Statistical analysis showed that patients carrying genetic variants in one or more of the five selected genes displayed a significantly lower response to glucocorticoids compared with subjects who had completely negative genetic results., Conclusions: The sequencing of multiple genes is of little help in the diagnostics of PFS and can often lead to results of uncertain interpretation, thus the clinically driven sequencing of single genes should remain the recommended approach. However, the presence of single or multiple genetic variants of uncertain significance, even if not allowing any specific diagnosis, correlated with a poorer response to glucocorticoids, possibly indicating a multifactorial subgroup of PFS with differential response to pharmacological treatment.

Glycogenosis type II, or Pompe Disease, is a lysosomal storage disease caused by the deficiency of acid alpha-glucosidase (GAA), leading to glycogen accumulation in muscles. A recombinant human GAA (rhGAA, Myozyme®) is currently used for enzyme replacement therapy. Despite its efficacy in most of patients, some of them show a diminished response to the treatment with rapidly progressive clinical deterioration, due to immuno-mediated enzyme inactivation. To demonstrate that Nanoparticles (NPs) could be profitably exploited to carry macromolecules, PLGA NPs loaded with rhGAA (GAA-NPs) were prepared by double emulsion solvent evaporation. Their surface morphology, particle size, zeta-potential and biochemical activity were assessed. "Pulse and chase" experiments were made by administrating GAA-NPs on patients' fibroblasts. Biochemical activity tests showed a more efficient cellular uptake of rhGAA loaded to NPs and a more significant stability of the enzyme (up to 7 days) in vitro, if compared to the same amount of rhGAA free enzyme. This data allows to envision in vivo experiments, in significant animal models, to further characterize lysosomal enzyme loaded-NPs' efficacy and toxicity.

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe₃O₄; size, 10-15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700-2,000 cm(-1) region. The precise PLGA IR-signature (1,750 cm(-1) centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs' fate in in vitro studies on cell cultures.

Congenital disorders of glycosylation (CDG) are genetic defects in protein and lipid glycosylation. PMM2-CDG is the most prevalent protein N-glycosylation disorder with more than 700 reported patients. Here we report on a large Italian family with four affected members and three mutations. Two young sisters are compound heterozygous for mutations p.Leu32Arg and p.Arg141His, while two paternal great-aunts are compound heterozygosity for p.Leu32Arg and p.Thr237Met. The latter association has not been reported before. The most severely affected member had in addition an ALG6 mutation known to exacerbate the phenotype of patients with PMM2-CDG., (© 2013 Elsevier B.V. All rights reserved.)

Periodic fever syndromes (PFSs) are a wide group of autoinflammatory diseases. Due to some clinical overlap between different PFSs, differential diagnosis can be a difficult challenge. Nowadays, there are no universally agreed recommendations for most PFSs, and near half of patients may remain without a genetic diagnosis even after performing multiple-gene analyses. Molecular analysis of periodic fevers' causative genes can improve patient quality of life by providing early and accurate diagnosis and allowing the administration of appropriate treatment. In this paper we focus our discussion on effective usefulness of genetic diagnosis of PFSs. The aim of this paper is to establish how much can the diagnostic system improve, in order to increase the success of PFS diagnosis. The mayor expectation in the near future will be addressed to the so-called next generation sequencing approach. Although the application of bioinformatics to high-throughput genetic analysis could allow the identification of complex genotypes, the complexity of this definition will hardly result in a clear contribution for the physician. In our opinion, however, to obtain the best from this new development a rule should always be kept well in mind: use genetics only to answer specific clinical questions.

Nanomedicine is certainly one of the scientific and technological challenges of the coming years. In particular, biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted delivery of different agents, including recombinant proteins, plasmid DNA, and low molecular weight compounds. PLGA NPs present some very attractive properties such as biodegradability and biocompatibility, protection of drug from degradation, possibility of sustained release, and the possibility to modify surface properties to target nanoparticles to specific organs or cells. Moreover, PLGA NPs have received the FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, thus reducing the time for human clinical applications. This review in particular deals on surface modification of PLGA NPs and their possibility of clinical applications, including treatment for brain pathologies such as brain tumors and Lysosomal Storage Disorders with neurological involvement. Since a great number of pharmacologically active molecules are not able to cross the Blood-Brain Barrier (BBB) and reach the Central Nervous System (CNS), new brain targeted polymeric PLGA NPs modified with glycopeptides (g7- NPs) have been recently produced. In this review several in vivo biodistribution studies and pharmacological proof-of evidence of brain delivery of model drugs are reported, demonstrating the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. Moreover, another relevant development of NPs surface engineering was achieved by conjugating to the surface of g7-NPs, some specific and selective antibodies to drive NPs directly to a specific cell type once inside the CNS parenchyma.

Lysosomes are not only degrading organelles but also involved in other critical cellular processes. In addition, active lysosomal glycohydrolases have been detected in an extra-lysosomal compartment: the presence of glycohydrolases on the plasma membrane (PM) has been widely demonstrated, and a possible role on the modification of the cell surface glycosphingolipids (GSL) participating in the modulation of cell functions such as cell-to-cell interactions and signal transduction pathways has been proposed. On this basis, the coordinated expression of lysosomal glycohydrolases and their translocation to the PM appear to be crucial for many cellular events. In this paper, we report evidence for the existence of a coordinated mechanism regulating the expression/activity of both lysosomal and PM-associated glycohydrolases. We show that the over-expression of the acidic glycohydrolase β-hexosaminidase α-subunit in mouse NIH/3T3 fibroblasts induces the increased expression of the Hex β-subunit necessary to form the active isoenzyme dimers as well as of other glycohydrolases participating in the GSL catabolism, such as β-galactosidase and β-glucocerebrosidase. More interestingly, this regulatory effect was also extended to the PM-associated hydrolases. In addition, transfected cells displayed a rearrangement of the GSL expression pattern that cannot be simply explained by the increased activity of a single enzyme. These observations clearly indicate that the expression level of metabolically related glycohydrolases is regulated in a coordinated manner and this regulation mechanism also involves the PM-associated isoforms.

In recent years, accumulating experimental evidence supports the notion that diabetic patients may greatly benefit from cell-based therapies, which include the use of adult stem and/or progenitor cells. In particular, mesenchymal stem cells and the circulating pool of endothelial progenitor cells have so far been the most studied populations of cells proposed for the treatment of vascular complications affecting diabetic patients. We review the evidence supporting their use in this setting, the therapeutic benefits that these cells have shown so far as well as the challenges that cell-based therapies in diabetic complications put out.

Hypertrophic cardiomyopathy (HCM) is the most frequent autosomal dominant genetic heart muscle disease and the most common cause of sudden cardiac death in young people (under 30 y of age), who are often unaware of their underlying condition. Genetic screening is now considered a fundamental tool for clinical management of HCM families. However, the high genetic heterogeneity of HCM makes genetic screening very expensive. Here, we propose a new high-throughput genotyping method based on a HCM 96-well sequencing plate for the analysis of 8 of the most frequent HCM-causing sarcomeric genes by automating several processes required for direct sequencing, using a commercially available robotic systems and routinely used instruments. To assess the efficiency of the robot-assisted method, we have analyzed the entire coding sequence and flanking intronic sequences of the 8 sarcomeric genes in samples from 18 patients affected by HCM and their relatives, which revealed 9 different mutations, 3 of which were novel. The automated, robot-assisted assembling of polymerase chain reaction, purification of polymerase chain reaction products, and assembly of sequencing reactions resulted in a substantial saving of time, reagent costs, and reduction of human errors, and can therefore be proposed as a primary strategy for mutation identification in HCM genetic screening in many medical genetic laboratories.

In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples.

The presence of the blood-brain barrier (BBB) makes extremely difficult to develop efficacious strategies for targeting contrast agents and delivering drugs inside the Central Nervous System (CNS). To overcome this drawback, several kinds of CNS-targeted nanoparticles (NPs) have been developed. In particular, we proposed poly-lactide-co-glycolide (PLGA) NPs engineered with a simil-opioid glycopeptide (g7), which have already proved to be a promising tool for achieving a successful brain targeting after i.v. administration in rats. In order to obtain CNS-targeted NPs to use for in vivo imaging, we synthesized and administrated in mice PLGA NPs with double coverage: near-infrared (NIR) probe (DY-675) and g7. The optical imaging clearly showed a brain localization of these novel NPs. Thus, a novel kind of NIR-labeled NPs were obtained, providing a new, in vivo detectable nanotechnology tool. Besides, the confocal and fluorescence microscopy evidences allowed to further confirm the ability of g7 to promote not only the rat, but also the mouse BBB crossing.

Background: Inherited mtDNA depletion syndromes (MDS) are a group of severe mitochondrial disorders resulting from defects in nucleus-encoded factors and often associated with severe or fatal liver failure., Patient: In this article, we describe the case of an 18-month-old patient with recurrent hypoketotic hypoglycaemia and fatal hepatic dysfunction with liver mtDNA depletion., Methods: The assessment of mtDNA copy number was performed on leucocytes, liver and muscle biopsy by Quantitative Real Time PCR and total RNA from liver biopsy was used as a template to amplify the cDNA of the POLG1 gene., Results: Sequence analysis identified two previously undescribed mutations (1868T>G and 2263A>G) located in the gene coding the catalytic subunit of mitochondrial DNA polymerase gamma (POLG), predicting an L623W and K755E amino acid change, respectively. Both mutations were located in the highly conserved linker region of the protein and were absent in more than 200 healthy unrelated control subjects. The identification of these two mutations allowed us to perform genetic counselling and prenatal diagnosis., Conclusion: Our data further expand the spectrum of POLG1 gene mutations and the unique phenotype reported (late onset isolated liver disease without lactic acidosis) increase the variability of clinical presentations associated with mutations in this gene.

We report a 12-year-old patient with growth retardation, exercise intolerance, lactic acidosis (increasing after exercise) and autoimmune polyendocrinopathy type 2. Muscle biopsy shows abundant COX-negative fibers, subsarcolemmal mitochondrial aggregates and markedly reduced activities of all respiratory chain complexes. Genetic analysis identified two new cosegregating mutations in Met-tRNA (m.4415A>G) and Cox III (m.9922A>C), located in highly conserved regions of MtDNA. Both the mutations are heteroplasmics in multiple patients' tissues. Single-muscle fiber analysis showed significantly higher levels of both the mutations in COX-negative than in normal fibers. In addition, a possible link between the mitochondrial dysfunction and the autoimmune disease is suggested.

Objective: The aim of this study was to provide a tool based on CDT measurements for a diagnostic approach to identify patients affected by congenital disorders of glycosylation (CDG) in a selected population., Design and Methods: Serum carbohydrate-deficient transferrin (CDT) of pediatric and adult patients (a total of 168 individuals) with neurological symptoms was analyzed. Abnormal results were confirmed by HPLC analysis and by enzymatic and molecular studies., Results: We found 6 patients (3.8%) with abnormal serum CDT; only two of them (1.9%) showed increased amounts of disialo and asialo with HPLC analysis and were classified as CDG-Ia, the most frequent form of CDG, due to a deficiency of the phosphomannomutase enzyme., Conclusions: The CDT quantitative immunoturbidimetric procedure is a useful tool for CDG screening. HPLC analysis, however, permitted the correct identification of asialo and disialo transferrin concentrations.

In an effort to develop an encapsulated cell-based system to deliver arylsulfatase A (ARSA) to the central nervous system of metachromatic leukodystrophy (MLD) patients, we engineered C2C12 mouse myoblasts with a retroviral vector containing a full-length human ARSA cDNA and evaluated the efficacy of the recombinant secreted enzyme to revert the MLD phenotype in oligodendrocytes (OL) of the As2-/- mouse model. After transduction, C2C12 cells showed a fifteen-fold increase in intracellular ARSA activity and five-fold increase in ARSA secretion. The secreted hARSA collected from transduced cells encapsulated in polyether-sulfone polymer, was taken up by enzyme-deficient OL derived from MLD mice and normally sorted to the lysosomal compartment, where transferred enzyme reached 80% of physiological levels, restoring the metabolism of sulfatide. To evaluate whether secreted enzyme could restore metabolic function in the brain, encapsulated cells and secreted ARSA were shown to be stable in CSF in vitro. Further, to test cell viability and enzyme release in vivo, encapsulated cells were implanted subcutaneously on the dorsal flank of DBA/2J mice. One month later, all retrieved implants released hARSA at rates similar to unencapsulated cells and contained well preserved myoblasts, demonstrating that encapsulation maintains differentiation of C2C12 cells, stable transgene expression and long-term cell viability in vivo. Thus, these results show the promising potential of developing an ARSA delivery system to the CNS based on the use of a polymer-encapsulated transduced xenogenic cell line for gene therapy of MLD.

The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC ). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex alpha-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells.

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.

Glycogenosis type 2 is an autosomal recessive glycogen storage disorder caused by deficiency of lysosomal acid alpha-glucosidase. Different phenotypes are recognized. The authors describe two children affected by the late infantile form; both presented terminal hyperthermia not caused by infections. Autopsy performed in one case showed diffuse glycogen storage in the CNS neurons. In light of current interest in enzyme replacement therapy, this finding casts some doubt on how effective enzyme replacement therapy will be unless it can be targeted directly into the CNS.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease, caused by deficiency of arylsulfatase A (ASA), that manifests primarily in the white matter of the nervous system. Currently, no specific treatment exists that will reverse its fatal outcome. Replacement therapy has been hampered by the blood-brain barrier (BBB). To circumvent this problem we designed an ex vivo gene therapy strategy that includes the retrovirus-mediated ASA transduction of cells, such as activated lymphocytes, that are able to traverse the BBB or other membranes of the CNS. For this purpose, two recombinant retroviruses based on the pLXSN vector were produced, containing the wild-type ASA cDNA or a pseudodeficiency ASA cDNA, which encodes a smaller enzyme with normal activity. After transduction, ASA activity increased more than 100-fold in fibroblasts from an MLD patient. Furthermore, ASA-transduced MLD PBLs expressed 30 times higher ASA activity when compared with control PBLs. Moreover, cell culture experiments demonstrated that transduced fibroblasts could efficiently transfer ASA to deficient cells across a transwell barrier, whereas transduced MLD lymphocytes could transfer ASA to deficient fibroblasts only by direct cell-to-cell contact. Finally, ASA was taken up by normal oligodendrocytes and Schwann cells, the target myelinating glial cells for therapy in MLD. These data suggest possible short-term strategies for transfer of ASA into the CNS via transduced autologous cells while long-term strategies, related to autologous transduced bone marrow transplant, take effect in patients.