An Association of PTPN11 and SHOX Mutations in a Male Presenting With Syndromic Growth Failure

In children with genetic syndromes, short stature is frequently a characteristic feature that, when associated with other specific manifestations, significantly aids in clinical diagnosis. In this report, an atypical case of Noonan syndrome (NS) in a 5.5-year-old child with mesomelic short stature is described. Genetic tests revealed two different mutations in this child. As expected in an NS case, a mutation in PTPN11 gene related to the RAS/MAPK signal transduction pathway was identified. Moreover, a mutation in the SHOX gene that was able to cause disproportionate short stature was detected. A clinical picture of NS with mesomelic short stature makes the diagnosis even more difficult as haploinsufficiency and complete loss of function of SHOX gene are associated with the typical differentiation and proliferation of chondrocytes, leading to mesomelic appearance. This case exemplifies the difficulties that can be encountered in achieving proper diagnoses for children with syndromic diseases and highlights the role of genetic tests in identifying final diagnoses in these patient

Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial (NCT03269071, EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale >= 6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients

Myocardial gene expression of osteopontin is higher in idiopathic than ischemic end-stage dilated cardiomyopathy

Purpose: The morphological and molecular features of cardiac remodelling are similar in idiopathic (DCM) as well as ischemic (ICM) human end-stage cardiomyopathy. However, the hallmarks of cardiac remodelling typical of DCM may be helpful to identify new treatment targets. Osteopontin (OPN), a phosphoglycoprotein of cardiac extracellular matrix, is an emerging mediator of cardiac inflammation and fibrosis in failing hearts. We have investigated whether the myocardial levels of OPN were affected by etiology of heart failure in the presence of similar left ventricular ejection fraction (LVEF). Methods: mRNA and protein levels of OPN were measured in LV samples from failing DCM (n=8; age: <50yrs; LVEF%=17.5±3; LVEDV=305.5±110ml) and ICM patients (n=8; age: <50yrs; LVEF%=19.5±5.2; LVEDV=270±97 ml) undergoing cardiac transplantation. All patients received conventional therapy for HF and underwent to cardiac function evaluation. As control (C), atrial samples of age and sex matched normal subjects (LVEF% ≥50) were analyzed. Real-time PCR analysis was carried out to measure OPN gene expression and data were normalized to three genes (RPS4X, eEF1a, RPL13a). The protein levels of OPN were assessed by enzyme immunometric assay. Results: Even though the extent of interstitial fibrosis in ICM was higher than DCM, OPN mRNA was significantly increased in DCM compared to C and ICM patients (C: 2.2±0.3; DCM: 31.3±7.4; ICM: 2.7±1.1, p=0.0004 C vs DCM and p=0.0002 DCM vs ICM). A similarly trend was observed for OPN cardiac protein concentration (C: 1.127±0.26; DCM: 1.29±0.22; ICM: 1.00±0.077 ng/ml). Conclusion: We have detected higher levels of OPN gene expression in LV samples of DCM rather than ICM failing hearts in the presence of similar LVEF. Our data suggest the new role of OPN as biomarker of myocardial remodelling typical of DCM independently of the fibrosis degree

Transcriptome profiling of natriuretic peptide system in cardiac tissue of patients with idiopathic or ischemic end-stage dilated cardiomyopathy

Purpose: Circulating levels of Natriuretic Peptides (NPs) play a role as reliable biomarkers of disease stage in patients with heart failure (HF). However, while several studies are present in literature about their circulating levels, only few data on the RNA expression have been reported and it is still debated whether the myocardial levels of NPs change in relation to etiology of cardiomyopathy in patients with similar left ventricular (LV) function. The aim of the study was to analyze the transcriptome profiling of NPs system in LV tissue of patients affected by idiopathic (DCM) or ischemic (ICM) end-stage dilated cardiomyopathy undergoing cardiac transplantation. Methods: Transcriptomic profile of NP system and of relative reference genes (RPS4X, eEF1a, RPL13a) was measured in LV myocardium of DCM (n=8; age: <50yrs; LVEF%=17.5±3; LVEDV=305.5±110ml) and ICM pts (n=8; age: <50yrs; LVEF%=19.5±5.2; LVEDV=270±97 ml) with similar cardiac function by Real-Time PCR analysis. All patients received similar medications and cardiac function was assessed by echocardiography. As control (C, n=5) atrial cardiac tissue samples of age and sex matched subjects with LVEF% ≥50 were analyzed. Results: All myocardial NPs resulted expressed in both DCM and ICM patients, yet BNP was the main expressed NP. In particular, ANP (DCM:0.445±0.26; ICM:0.077±0.05) and BNP (DCM:9.89±1.92; ICM:3.29±1.4, p=0.007) resulted higher in DCM compared to ICM, while CNP was more higher in ICM, even though not significantly (DCM:0.864±0.4; ICM:1.85±0.99). All NP receptors were expressed in both groups. Even though BNP and CNP levels were higher in both failing hearts compared to C, between the NP receptors only NPR-B resulted increased in DCM (p<0,0001 C vs. DCM). Significant correlations were observed between BNP and NPR-A, NPR-B and NPR-C (p=0.022 and p=0.044, p<0.0001 respectively). Conclusions: Our results show, in the presence of similar decay of the LV function, an activation of the entire NP system reporting a different involvement in cardiac tissue of ICM/DCM patients and underlining a different trend for CNP mRNA expression. Even though more studies are necessary to better explain the role of CNP in the setting of cardiomyopathies, these findings support the future use of CNP as a new target in the treatment of ischemic disorders, thanks to its protective effects. Moreover its different transcriptomic levels, depending on the aetiology, together with the activation of its specific receptor, NPR-B, could be used in future clinical application as important diagnostic tool

An Association of PTPN11 and SHOX Mutations in a Male Presenting With Syndromic Growth Failure

In children with genetic syndromes, short stature is frequently a characteristic feature that, when associated with other specific manifestations, significantly aids in clinical diagnosis. In this report, an atypical case of Noonan syndrome (NS) in a 5.5-year-old child with mesomelic short stature is described. Genetic tests revealed two different mutations in this child. As expected in an NS case, a mutation in PTPN11 gene related to the RAS/MAPK signal transduction pathway was identified. Moreover, a mutation in the SHOX gene that was able to cause disproportionate short stature was detected. A clinical picture of NS with mesomelic short stature makes the diagnosis even more difficult as haploinsufficiency and complete loss of function of SHOX gene are associated with the typical differentiation and proliferation of chondrocytes, leading to mesomelic appearance. This case exemplifies the difficulties that can be encountered in achieving proper diagnoses for children with syndromic diseases and highlights the role of genetic tests in identifying final diagnoses in these patients

Myocardial Expression Analysis of Osteopontin and Its Splice Variants in Patients Affected by End-Stage Idiopathic or Ischemic Dilated Cardiomyopathy.

Osteopontin (OPN) is a phosphoglycoprotein of cardiac extracellular matrix and it is still poorly defined whether its expression changes in failing heart of different origin. The full-length OPN-a and its isoforms (OPN-b, OPN-c) transcriptomic profile were evaluated in myocardium of patients with dilated or ischemic cardiomyopathy (DCM n = 8; LVEF% = 17.5±3; ICM n = 8; LVEF% = 19.5±5.2) and in auricle of valvular patients (VLP n = 5; LVEF%≥50), by Real-time PCR analysis. OPN-a and thrombin mRNA levels resulted significantly higher in DCM compared to ICM patients (DCM:31.3±7.4, ICM:2.7±1.1, p = 0.0002; DCM:19.1±4.9, ICM:5.4±2.2, p = 0.007, respectively). Although both genes' mRNA levels increased in patients with LVEF50%, a significant increase in OPN (p = 0.0004) and thrombin (p = 0.001) expression was observed only in DCM. In addition, a correlation between OPN-a and thrombin was found in patients with LVEF<50% (r = 0.6; p = 0.003). The mRNA pattern was confirmed by OPN-a cardiac protein concentration (VLP:1.127±0.26; DCM:1.29±0.22; ICM:1.00±0.077 ng/ml). The OPN splice variants expression were detectable only in ICM (OPN-b: 0.357±0.273; OPN-c: 0.091±0.033) and not in DCM patients. A significant correlation was observed between collagen type I, evaluated by immunohistochemistry analysis, and both OPN-a mRNA expression (r = 0.87, p = 0.002) and OPN protein concentrations (r = 0.77, p = 0.016). Concluding, OPN-a and thrombin mRNA resulted dependent on the origin of heart failure while OPN-b and OPN-c highlighted a different expression for DCM and ICM patients, suggesting their correlation with different clinical-pathophysiological setting

Dipyridamole-induced C-type natriuretic peptide mRNA overexpression in a minipig model of pacing-induced left ventricular dysfunction.

Dipyridamole (DP) restores ischemic tissue blood flow stimulating angiogenesis in eNOS-dependent pathways. C-type natriuretic peptide (CNP) is expected to mimic the migration-stimulatory effect of NO via a cGMP-dependent mechanism. Aim of this study was to assess the role of concomitant treatment with DP on CNP levels in blood and myocardial tissue of minipigs with left ventricular dysfunction (LVD) induced by pacing at 200bpm in the right ventricular apex. Minipigs with DP therapy (DP+, n=4) or placebo (DP-, n=4) and controls (C-SHAM, n=4) underwent 2D-EchoDoppler examination and blood collection before and after 4 weeks of pacing, when cardiac tissue was collected. Histological/immunohistochemical analyses were performed. CNP levels were determined by radioimmunoassay; cardiac CNP, BNP, natriuretic receptors expression by Real-Time PCR. After pacing, cardiac parameters resulted less impaired in DP+ compared to DP-. Histological sections presented normal morphology while the arteriolar density resulted: C-SHAM: 9.0±1.2; DP-: 4.9±0.3; DP+: 6.5±0.6number/mm(2); C-SHAM vs DP- and DP+ p=0.004, p=0.04, respectively. CNP mRNA resulted lower in DP- compared to C-SHAM and DP+ as well as NPR-B (p=0.011, DP- vs DP+). Both NPR-A/NPR-C mRNA expressions were significantly (p<0.001) lower both in DP- and DP+ compared to C-SHAM. BNP mRNA was higher in LVD. CNP plasma levels showed a similar trend with respect to gene expression (C-SHAM: 30.5±15; DP-: 18.6±5.5; DP+: 21.2±4.7pg/ml). These data suggest that DP may serve as a preconditioning agent to increase the protective CNP-mediated endocrine response in LVD. This response, mediated by its specific receptor NPR-B, may offer new insights into molecular targets for treatment of LVD

Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+ Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy

According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+ cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 106 of CD133+ cells (range 2.85 × 106–30.84 × 106), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133+ cells of 90,60% (range 81,40%–96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106 cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial)