Intracerebroventricular transplantation of human iPSC-derived neural stem cells (hiPSC-NSCs) into neonatal mice.

Human neural stem cells (hNSCs) hold great promises for the development of cell-based therapies for neurodegenerative diseases, given their capability to provide immunomodulatory and trophic support and to replace, to a limited extent, damaged, or lost cells. Human NSCs are under clinical evaluation for the treatment of several neurodegenerative diseases. Still, issues related to the large-scale production of clinical-grade fetal hNSCs and their allogeneic nature-requiring immunosuppressive regimens-have hampered their full exploitation as therapeutics. NSCs derived from human induced pluripotent stem cells (hiPSCs) provide a valuable alternative to fetal hNSCs since they can be generated from autologous or HLA-matched donors expanded for large-scale clinical-grade production, and are amenable for gene addition/gene editing strategies, thus potentially addressing CNS diseases of genetic origin. The prospective use of hiPSC-derived NSCs (hiPSC-NSCs) for CNS-directed therapies demands a careful evaluation of the efficacy and safety of these cell populations in animal models. Here, we describe a protocol for the transplantation and phenotypical characterization of hiPSC-NSCs in neonatal immunodeficient mice. This protocol is relevant to assessing the safety and the efficacy of hiPSC-NSC transplantation to target early-onset neurodegenerative or demyelinating CNS diseases.

Delivery Platforms for CRISPR/Cas9 Genome Editing of Glial Cells in the Central Nervous System.

Glial cells (astrocytes, oligodendrocytes, and microglia) are emerging as key players in several physiological and pathological processes of the central nervous system (CNS). Astrocytes and oligodendrocytes are not only supportive cells that release trophic factors or regulate energy metabolism, but they also actively modulate critical neuronal processes and functions in the tripartite synapse. Microglia are defined as CNS-resident cells that provide immune surveillance; however, they also actively contribute to shaping the neuronal microenvironment by scavenging cell debris or regulating synaptogenesis and pruning. Given the many interconnected processes coordinated by glial cells, it is not surprising that both acute and chronic CNS insults not only cause neuronal damage but also trigger complex multifaceted responses, including neuroinflammation, which can critically contribute to the disease progression and worsening of symptoms in several neurodegenerative diseases. Overall, this makes glial cells excellent candidates for targeted therapies to treat CNS disorders. In recent years, the application of gene editing technologies has redefined therapeutic strategies to treat genetic and age-related neurological diseases. In this review, we discuss the advantages and limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing in the treatment of neurodegenerative disorders, focusing on the development of viral- and nanoparticle-based delivery methods for in vivo glial cell targeting.

The long and winding road to target protein misfolding in cardiovascular diseases.

BACKGROUND: In the last decades, cardiovascular diseases (CVD) have remained the first leading cause of mortality and morbidity in the world. Although several therapeutic approaches have been introduced in the past, the development of novel treatments remains an important research goal, which is hampered by the lack of understanding of key mechanisms and targets. Emerging evidences in recent years indicate the involvement of misfolded proteins aggregation and the derailment of protein quality control in the pathogenesis of cardiovascular diseases. Several potential interventions targeting protein quality control have been translated from the bench to the bedside to effectively employ the misfolded proteins as promising therapeutic targets for cardiac diseases, but with trivial results. DESIGN: In this review, we describe the recent progresses in preclinical and clinical studies of protein misfolding and compromised protein quality control by selecting and reporting studies focusing on cardiovascular diseases including cardiomyopathies, cardiac amyloidosis, atherosclerosis, atrial fibrillation and thrombosis. RESULTS: In preclinical models, modulators of several molecular targets (eg heat shock proteins, unfolded protein response, ubiquitin protein system, autophagy and histone deacetylases) have been tested in various conditions with promising results although lacking an adequate transition towards clinical setting. CONCLUSIONS: At present, no therapeutic strategies have been reported to attenuate proteotoxicity in patients with CVD due to a lack of specific biomarkers for pinpointing upstream events in protein folding defects at a subclinical stage of the diseases requiring an intensive collaboration between basic scientists and clinicians.

Current and future circulating biomarkers for cardiac amyloidosis.

Cardiac amyloidosis (CA) comprises a heterogeneous group of medical conditions affecting the myocardium. It presents with proteinaceous infiltration with variable degrees of severity, prevalence and evolution. Despite this heterogeneity, erroneous protein folding is the common pathophysiologic process, yielding the formation of a single misfolded protein (monomer) that progressively evolves and ultimately forms amyloid fibers. Additionally, by seeding out from the organs of origin, intermediates called oligomers metastasize and restart the process. Such self-echoing behavior makes the secondary affected organs as important as the primary ones. Unfortunately, CA can be clinically challenging and only suggestive in a late stage of its natural history, leaving a narrow therapeutic time window available. In light of the evolutionary nature of amyloidosis, here, we propose a new classification of the currently used biomarkers based on time stages with different specificity and applicability across CA subtypes. Early markers (free light chains, serum amyloid A, ß2-microglobulin, osteopontin and osteoprotegerin) can be employed for disease detection. Intermediate markers [soluble suppression of tumorigenicity 2 (sST-2), midregional proadrenomedullin (MR-proADM), von Willebrand factor (vWF), hepatocyte growth factor (HGF), matrix metalloproteinases (MMPs) and tissue inhibitor metalloproteinases (TIMPs)] can provide information on the biological mechanisms of myocardial damage. As in heart failure, late-stage biomarkers (troponins and natriuretic peptides) can help clinicians with prognosis and therapeutic response evaluation in CA. Such findings have generated a remarkable foundation for our current knowledge on CA. Nevertheless, we envision a future class of biomarkers targeted at upstream events capable of detecting folding defects, which will ultimately expand the therapeutic window.

Fungal Endocarditis Due to Aspergillus oryzae: The First Case Reported in the Literature.

Infective endocarditis (IE) is a severe disease with high mortality and morbidity. Prosthetic valve endocarditis is a life-threatening complication which can occur in less than 10% of patients with valve prosthesis. A fungal etiology of IE is rare and accounts for only 2-4% of all case of endocarditis, but is associated with a higher mortality and morbidity. Herein is reported the first case of fungal endocarditis of aortic valve prosthesis due to Aspergillus oryzae in a 67-year-old caucasian man who nine years previously underwent mitral and aortic valve replacement with mechanical prostheses, and tricuspid annuloplasty for acute IE due to Enterococcus spp. Seven months previously, the patient also underwent a redo cardiac procedure to replace a mitral valve prosthesis with a new mechanical device due to a leakage. Aspergillus oryzae showed impressive growth with strong and unexpected virulence in both local and systemic settings.

Prosthetic valve endocarditis: predictors of early outcome of surgical therapy. A multicentric study.

OBJECTIVES: Prosthetic valve endocarditis (PVE) is an uncommon yet dreadful complication in patients with prosthetic valves that requires a distinct analysis from native valve endocarditis. The present study aims to investigate independent risk factors for early surgical outcomes in patients with PVE. METHODS: A retrospective cohort study was conducted in 8 Italian Cardiac Surgery Units from January 2000 to December 2013 by enrolling all PVE patients undergoing surgical treatment. RESULTS: A total of 209 consecutive patients were included in the study. During the study period, the global rate of surgical procedures for PVE among all operations for isolated or associated valvular disease was 0.45%. Despite its rarity this percentage increased significantly during the second time frame (2007-2013) in comparison with the previous one (2000-2006): 0.58% vs 0.31% (P < 0.001). Intraoperative and in-hospital mortality rates were 4.3% and 21.5%, respectively. Logistic regression analysis identified the following factors associated with in-hospital mortality: female gender [odds ratio (OR) = 4.62; P < 0.001], shock status (OR = 3.29; P = 0.02), previous surgical procedures within 3 months from the treatment (OR = 3.57; P = 0.009), multivalvular involvement (OR = 8.04; P = 0.003), abscess (OR = 2.48; P = 0.03) and urgent surgery (OR = 6.63; P < 0.001). CONCLUSIONS: Despite its rarity, PVE showed a significant increase over time. Up to now, in-hospital mortality after surgical treatment still remains high (>20%). Critical clinical presentation and extension of anatomical lesions are strong preoperative predictors for poor early outcome.

Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

Aß Amyloid Pathology Affects the Hearts of Patients With Alzheimer's Disease: Mind the Heart.

BACKGROUND: Individually, heart failure (HF) and Alzheimer's disease (AD) are severe threats to population health, and their potential coexistence is an alarming prospect. In addition to sharing analogous epidemiological and genetic profiles, biochemical characteristics, and common triggers, the authors recently recognized common molecular and pathological features between the 2 conditions. Whereas cognitive impairment has been linked to HF through perfusion defects, angiopathy, and inflammation, whether patients with AD present with myocardial dysfunction, and if the 2 conditions bear a common pathogenesis as neglected siblings are unknown. OBJECTIVES: Here, the authors investigated whether amyloid beta (Aß) protein aggregates are present in the hearts of patients with a primary diagnosis of AD, affecting myocardial function. METHODS: The authors examined myocardial function in a retrospective cross-sectional study from a cohort of AD patients and age-matched controls. Imaging and proteomics approaches were used to identify and quantify Aß deposits in AD heart and brain specimens compared with controls. Cell shortening and calcium transients were measured on isolated adult cardiomyocytes. RESULTS: Echocardiographic measurements of myocardial function suggest that patients with AD present with an anticipated diastolic dysfunction. As in the brain, Aß40 and Aß42 are present in the heart, and their expression is increased in AD. CONCLUSIONS: Here, the authors provide the first report of the presence of compromised myocardial function and intramyocardial deposits of Aß in AD patients. The findings depict a novel biological framework in which AD may be viewed either as a systemic disease or as a metastatic disorder leading to heart, and possibly multiorgan failure. AD and HF are both debilitating and life-threatening conditions, affecting enormous patient populations. Our findings underline a previously dismissed problem of a magnitude that will require new diagnostic approaches and treatments for brain and heart disease, and their combination.

Late reoperations after acute aortic dissection repair: Single-center experience.

BACKGROUND: After repair of acute type A aortic dissection, aortic complications can develop, and reoperations might be necessary. In our retrospective study, we wanted to assess early and late outcomes in this cohort of patients. METHODS: From September 2005 to July 2012, 21 consecutive patients previously operated on for acute type A aortic dissection underwent 27 redo aortic surgical procedures. Indications for redo procedures were: enlargement of the false lumen in the residual aorta (18 events), severe aortic regurgitation with or without aortic root dilatation (8 events), suture dehiscence and pseudoaneurysm at the proximal or distal aortic graft anastomosis (5 events) or at the coronary button anastomosis in patients who previously underwent a Bentall procedure (1 patient). In all cases, total or partial cardiopulmonary bypass was used. Hypothermic cardiocirculatory arrest was needed in 22 (81%) procedures. RESULTS: Hospital mortality was 3.7% (1/27), reexploration for bleeding and paraplegia rates were 7.4% and 7.4%, respectively. Marfan patients received 3.2 procedures per patient vs. 1.5 in non-Marfan patients (p < 0.01). At a mean follow-up of 6.5 years, 2 aortic events occurred: 1 aortic death, and 1 additional aortic redo surgery. CONCLUSIONS: When procedures are carried out on elective basis, redo aortic surgery can be performed in all segments of the aorta with good early and late outcomes. Close lifelong clinical and radiological follow-up is mandatory. After repair of acute type A aortic dissection, Marfan patients are more prone to develop late complications, with a more rapid evolution.

Hsa-miR-15a and Hsa-miR-16-1 expression is not related to proliferation centers abundance and other prognostic factors in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) is the commonest leukemia in adults. Here, we aimed to evaluate hsa-miR-15a/hsa-miR-16-1 expression in CLL tissues by qPCR and correlate it with the other clinicopathological features and clinical outcome. 40 formalin-fixed paraffin-embedded (FFPE) lymph node samples obtained from CLL/SLL patients were classified into two categories, "PCs rich" and "typical." We found a significant common expression level of 4 miRNAs; however, we did not find any significant relationship between PCs presence and miRNAs expression. Moreover, neither the presence of 13q deletion nor the percentage of cells carrying the deletion strictly correlated with miRNAs expression levels, although a significant number of patients with 13q deletion presented hsa-miR-16-1-3p levels below the median value in normal samples (P < 0.05). Finally, although no correlation was found between the expression of each miRNA and other clinicopathological features (Ki67, CD38, ZAP70, and IGVH@ hypermutations), the OS curves showed a positive trend in patients with miRNAs downregulation, though not statistically significant. In conclusion, we showed for the first time that all miRNAs can be successfully studied in FFPE CLL tissues and that del13q and PCs richness do not strictly correspond to miRNAs downregulation; therefore, a specific evaluation may be envisaged at least in patients enrolled in clinical trials.