Brain cortical assessment by MRI in fetuses with left congenital diaphragmatic hernia.

OBJECTIVE: To evaluate fetal brain development using MRI (magnetic resonance imaging) in CDH (congenital diaphragmatic hernia). METHODS: 52 isolated left CDH and 104 control fetuses were imaged using MRI. Brain morphometry (Biparietal diameter-BPD, brain fronto-occipital diameter-BFOD, third ventricle, posterior ventricles, transcerebellar diameter-TCD, anteroposterior and craniocaudal cerebellar vermis diameter-AP and CC) and cortical structures (bilateral cingulate fissure-CF, insular fissure-IF, insular depth - ID) were compared with controls using Mann-Whitney test. RESULTS: Median gestational age at MRI (p = 0.95)and the median biparietal diameter (p = 0.737) were comparable. Among morphometric parameters, only the brain fronto-occipital diameter was significantly smaller in CDH (p = 0.001) and the third ventricle was significantly greater in CDH (<0.0001). Among cortical structures, the cingulate and insular fissures were significantly deeper in CDH fetuses (p < 0.0001) as the insular depth ID was smaller in CDH (p < 0.03). CONCLUSIONS: CDH fetuses have a smaller fronto-occipital diameter, reduced insular depth, deeper cingulate and insular fissure, and greater third ventricle width as compared to controls. These findings suggest that left CDH may have an impact on fetal brain development with an overall reduction in brain volume.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product.

BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

Fetal MRI assessment of mediastinal shift angle in isolated left congenital diaphragmatic hernia: A new postnatal survival predictive tool?

OBJECTIVE: To quantify the mediastinal shift angle (MSA) in fetuses with isolated left congenital diaphragmatic hernia (CDH) by magnetic resonance imaging and evaluate survival. METHOD: Fetuses from singleton pregnancies with isolated left CDH were matched for gestational age with controls without thoracic malformations. For all fetuses the MSA was determined by two operators and inter-operator variability and differences between cases and controls were investigated. For all cases total fetal lung volume (TFLV) was calculated and the correlation between MSA and TFLV was assessed, and its predictive value towards survival was determined. RESULTS: Thirty-four fetuses were included as cases and 42 as controls. The mean gestational age for assessment of CDH fetuses was 32 weeks (range 27-38). Twenty-four fetuses survived until discharge and 10 did not. There was an excellent inter-operator reliability for measuring the MSA and a significant difference between MSA in cases and controls. There was an inverse correlation between MSA values and survival, a correlation between TFLV and survival and an inverse correlation between MSA and TFLV. The area under the ROC curve for MSA in predicting survival was 0.931 (95% CI 0.851-1.000). CONCLUSION: The MSA measured late in gestation correlates with postnatal survival in patients with isolated left CDH.

Pooled human serum: A new culture supplement for bioreactor-based cell therapies. Preliminary results.

BACKGROUND: Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System. METHODS: Bone marrow aspirates were obtained from healthy adult individuals undergoing routine total hip replacement surgery and used to generate primary cultures in the bioreactor. HPL and ABS were tested as supplements to culture medium. Morphological observations, cytofluorimetric analysis, lactate and glucose level assessment were performed. RESULTS: ABS gave rise to both heterogeneous MSC and MPC population. About 95% of cells cultured in HPL showed a fibroblast-like morphology and typical mesenchymal surface markers, but MPCs were scarcely represented. DISCUSSION: The use of ABS appeared to sustain a large scale MSC production, as well as the recovery of a subset of MPCs, and resulted a suitable alternative to HPL in the cell generation based on the Quantum System.

Acute encephalopathy in the immune-compromised child: never forget toxoplasmosis.

Toxoplasma gondii is an opportunistic parasite, which very unusually may cause acute encephalitis in patients undergoing chemotherapy or hematopoietic stem cell transplant. The prognosis is usually dismal also because of late diagnosis, depending on the limited availability of specific diagnostic tools. An early diagnosis allows effective intervention with specific antibiotics, which may provide a chance for cure. We report 2 cases of cerebral toxoplasmosis in which the use of polymerase chain reaction on cerebrospinal fluid allowed a prompt diagnosis and specific therapy, which was followed by clinical response and negativization at follow-up studies of T. gondii genome on cerebrospinal fluid by polymerase chain reaction and by brain imaging.

Safety and efficacy of topiramate in neonates with hypoxic ischemic encephalopathy treated with hypothermia (NeoNATI).

BACKGROUND: Despite progresses in neonatal care, the mortality and the incidence of neuro-motor disability after perinatal asphyxia have failed to show substantial improvements. In countries with a high level of perinatal care, the incidence of asphyxia responsible for moderate or severe encephalopathy is still 2-3 per 1000 term newborns. Recent trials have demonstrated that moderate hypothermia, started within 6 hours after birth and protracted for 72 hours, can significantly improve survival and reduce neurologic impairment in neonates with hypoxic-ischemic encephalopathy. It is not currently known whether neuroprotective drugs can further improve the beneficial effects of hypothermia. Topiramate has been proven to reduce brain injury in animal models of neonatal hypoxic ischemic encephalopathy. However, the association of mild hypothermia and topiramate treatment has never been studied in human newborns. The objective of this research project is to evaluate, through a multicenter randomized controlled trial, whether the efficacy of moderate hypothermia can be increased by concomitant topiramate treatment. METHODS/DESIGN: Term newborns (gestational age ≥ 36 weeks and birth weight ≥ 1800 g) with precocious metabolic, clinical and electroencephalographic (EEG) signs of hypoxic-ischemic encephalopathy will be randomized, according to their EEG pattern, to receive topiramate added to standard treatment with moderate hypothermia or standard treatment alone. Topiramate will be administered at 10 mg/kg once a day for the first 3 days of life. Topiramate concentrations will be measured on serial dried blood spots. 64 participants will be recruited in the study. To evaluate the safety of topiramate administration, cardiac and respiratory parameters will be continuously monitored. Blood samplings will be performed to check renal, liver and metabolic balance. To evaluate the efficacy of topiramate, the neurologic outcome of enrolled newborns will be evaluated by serial neurologic and neuroradiologic examinations. Visual function will be evaluated by means of behavioural standardized tests. DISCUSSION: This pilot study will explore the possible therapeutic role of topiramate in combination with moderate hypothermia. Any favourable results of this research might open new perspectives about the reduction of cerebral damage in asphyxiated newborns.

Comparison of mediastinal shift angles obtained with ultrasound and magnetic resonance imaging in fetuses with isolated left sided congenital diaphragmatic hernia.

OBJECTIVES: To compare ultrasound (US) and magnetic resonance imaging (MRI) in the assessment of mediastinal shift angles (MSAs) in fetuses affected by isolated left congenital diaphragmatic hernia (CDH). The use of MRI-MSA and US-MSA as prognostic factor for postnatal survival in fetal left CDH was also explored. METHODS: This was an observational study of 29 fetuses with prenatally diagnosed isolated left CDH, assessed with both US and MRI examinations between January 2015 and December 2018. The US-MSA measurements performed within 2 weeks from the MRI assessment were considered for the analysis. The primary outcome was the postnatal survival rate. RESULTS: No significant difference between US and MRI MSAs was detected (p = .419). Among the 29 cases, there were 21 alive infants, for an overall postnatal survival rate of 72.41%. After stratifying for postnatal survival, the best cutoffs with the highest discriminatory power in terms of sensibility and specificity were 42.1° for the US-MSA and 39.1° for the MRI-MSA. The performance of MRI-MSA in predicting postnatal survival was close to that of US-MSA in terms of sensitivity (62.5 versus 50.0%), specificity (80.9 versus 90.5%), positive predictive value (55.6 versus 66.7%), negative predictive value (85.0 versus 82.6%) and accuracy (75.9 versus 79.3%). There was no statistically significant difference between the two modalities (p > .05 for all). CONCLUSIONS: MRI and US can be interchangeably used for the assessment of MSA in prenatally diagnosed isolated left CDH. Moreover, MSA measured by both US and MRI was confirmed to be correlated with perinatal outcome in terms of survival.

Morphometric Analysis of Brain in Newborn with Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) is a severe pediatric disorder with herniation of abdominal viscera into the thoracic cavity. Since neurodevelopmental impairment constitutes a common outcome, we performed morphometric magnetic resonance imaging (MRI) analysis on CDH infants to investigate cortical parameters such as cortical thickness (CT) and local gyrification index (LGI). By assessing CT and LGI distributions and their correlations with variables which might have an impact on oxygen delivery (total lung volume, TLV), we aimed to detect how altered perfusion affects cortical development in CDH. A group of CDH patients received both prenatal (i.e., fetal stage) and postnatal MRI. From postnatal high-resolution T2-weighted images, mean CT and LGI distributions of 16 CDH were computed and statistically compared to those of 13 controls. Moreover, TLV measures obtained from fetal MRI were further correlated to LGI. Compared to controls, CDH infants exhibited areas of hypogiria within bilateral fronto-temporo-parietal labels, while no differences were found for CT. LGI significantly correlated with TLV within bilateral temporal lobes and left frontal lobe, involving language- and auditory-related brain areas. Although the causes of neurodevelopmental impairment in CDH are still unclear, our results may suggest their link with altered cortical maturation and possible impaired oxygen perfusion.

MRI and DWI: feasibility of DWI and ADC maps in the evaluation of placental changes during gestation.

OBJECTIVE: To establish if a correlation exists between apparent diffusion coefficient (ADC) values, obtained by diffusion-weighted imaging (DWI), and placental aging. METHOD: The study is divided into a retrospective phase and a prospective one.In the first phase, 145 pregnant women underwent fetal magnetic resonance imaging (MRI) for suspected disorders in several organs. We performed DWI (b value 0, 200 and 700 s/mm(2)) in all the fetuses, evaluating the patients in whom the whole placenta was visible.In the prospective phase, 50 women (52 fetuses) underwent MRI. We performed, in the same patient, two echo-planar sequences with b values of 0, 200 and 700, and 50, 200 and 700 s/mm(2), including the whole placenta.The ADC maps were calculated for all fetuses, divided into three groups based on gestational age (GA): group I: 20-26 weeks' gestation, II: 27-33, III: 34-40. RESULTS: In the retrospective phase, ADC values had a range from 1 to 2.4 mm(2)/s, showing a significant correlation between ADC values and GA.ADC values obtained by DWI with b value 0, 200 and 700 s/mm(2) had a range from 0.8 to 2.5 mm(2)/s, with an inverse correlation between ADC values and GA, whereas the ADC values with b value 50, 200 and 700 s/mm(2) did not show any statistical correlation (range: 1.5-1.7 mm(2)/s). CONCLUSION: DWI with ADC maps can not be considered markers for placental aging because they are affected by perfusional and circulatory motion.

Fetal abdominal lymphangioma enhanced by ultrafast MRI.

Abdominal lymphangioma represents an uncommon congenital anomaly. Herein, we describe an unusual case, which on routine antenatal ultrasonography masqueraded as isolated ascites. Subsequent fetal MRI at 27 weeks' gestation implemented antenatal assessment of the lesion, prompting further investigations and straightforward postnatal treatment. Fetal MRI represents a valuable adjunct to antenatal ultrasonography for the evaluation of cystic abdominal masses.

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions - namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Early Diagnosis and Monitoring of Neurodegenerative Langerhans Cell Histiocytosis.

BACKGROUND: Neurodegenerative Langerhans Cell Histiocytosis (ND-LCH) is a rare, unpredictable consequence that may devastate the quality of life of patients cured from LCH. We prospectively applied a multidisciplinary diagnostic work-up to early identify and follow-up patients with ND-LCH, with the ultimate goal of better determining the appropriate time for starting therapy. METHODS: We studied 27 children and young adults with either ND-LCH verified by structural magnetic resonance imaging (MRI) (group 1) or specific risk factors for (diabetes insipidus, craniofacial bone lesions), but no evidence of, neurodegenerative MRI changes (group 2). All patients underwent clinical, neurophysiological and MRI studies. RESULTS: Seventeen patients had MRI alterations typical for ND-LCH. Nine showed neurological impairment but only three were symptomatic; 11 had abnormal somatosensory evoked potentials (SEPs), and five had abnormal brainstem auditory evoked potentials (BAEPs). MR spectroscopy (MRS) showed reduced cerebellar NAA/Cr ratio in nine patients. SEPs showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for predicting ND-LCH of 70.6% (95%CI, 44.0%-89.7%), 100% (69.2%-100%), 100% (73.5%-100%), and 66.7% (38.4%-88.2%), respectively. Repeated investigations in group 1 revealed increasingly abnormal EP parameters, or neurological examination, or both, in nine of fifteen patients while MRI remained unchanged in all but one patient. CONCLUSION: A targeted MRI study should be performed in all patients with risk factors for ND-LCH for early identification of demyelination. The combined use of SEPs and careful neurological evaluation may represent a valuable, low-cost, well-tolerated and easily available methodology to monitor patients from pre-symptomatic to symptomatic stages. We suggest a multidisciplinary protocol including clinical, MRS, and neurophysiological investigations to identify a population target for future therapeutic trials.

Chest fast MRI: an imaging alternative on pre-operative evaluation of Pectus Excavatum.

BACKGROUND: Standard imaging methods in evaluating chest wall deformities, such as Pectus Excavatum (PE) in paediatric and adolescent patients, include baseline 2-view chest radiography and chest CT scan. Only few studies to date investigated the value of fast MRIin the pre operative assessment of patient affected by PE. OBJECTIVE: To evaluate the efficacy of chest fast MRI in pre-operative management of patient affected by PE. To obtain the Haller Index (HI) and Asymmetry Index (AI) from chest fast MRI protecting patients from radiation exposure. MATERIALS AND METHODS: We analyzed the data of 42 consecutive patients with severe PE who underwent minimally invasive repair between March 2007 and March 2010. All 42 patients received chest fast MRI, but only the first 5 in view of the results, were studied also with chest ultrafast CT scan. In both examinations, data at the deepest point of the depression were collected. RESULTS: Severity indices of the deformity using HI and AI, collected from CT scan and fast MRI in the first 5 patients, were comparable. In the remaining 37 fast chest MRI offered good images of the chest wall deformities with no radiation exposure, detailing anatomical information such as displacement and rotation of the heart or great vessels anomalies. CONCLUSION: This study suggests the use of chest MRI in pre operative workup for patients with PE to obtain severity indices (Haller Index and Asymmetry Index avoiding radiation exposure to paediatric patients.

Exploring PTX3 expression in Sus scrofa cardiac tissue using RNA sequencing.

The prototypic long pentraxin PTX3 is a novel vascular inflammatory marker sharing similarities with the classic short pentraxin (C-reactive protein). PTX3 is rapidly produced and released by several cell types in response to local inflammation of the cardiovascular system. Plasma PTX3 levels are very low in normal conditions and increase in heart failure (HF) patients with advancing NYHA functional class, but its exact role during HF pathogenetic mechanisms is not yet established. No data about PTX3 cardiac expression in normal and pathological conditions are currently available, either in human or in large-size animals. Of the latter, the pig has a central role in "in vivo" clinical settings but its genome has not been completely sequenced and the PTX3 gene sequence is still lacking. The aim of this study was to sequence the PTX3 in Sus scrofa, whose sequence is not yet present in GenBank. Utilizing our knowledge of this sequence, PTX3 mRNA expression was evaluated in cardiac tissue of normal (n=6) and HF pigs (n=5), obtained from the four chambers. To sequence PTX3 gene in S. scrofa, the high homology between Homo sapiens and S. scrofa was exploited. Pig PTX3 mRNA was sequenced using polymerase chain reaction primers designed from human consensus sequences. The DNA, obtained from different RT-PCR reactions, was sequenced using the Sanger method. S. scrofa PTX3 mRNA, 1-336 bp, was submitted to GenBank (ID: GQ412351). The sequence obtained from pig cardiac tissue shared an 84% sequence identity with human homolog. The presence of PTX3 mRNA expression was detected in all the cardiac chambers sharing an increase after 3 weeks of pacing compared to controls (p=0.036 HF right atrium vs. N; p=0.022, HF left ventricle vs. N). Knowledge of the PTX3 sequence could be a useful starting point for future studies devoted to better understanding the specific role of this molecule in the pathogenesis of cardiovascular diseases.

Early predictors of juvenile sacroiliitis in enthesitis-related arthritis.

OBJECTIVE: To identify early predictors of sacroiliac (SI) involvement in a cohort of patients with enthesitis-related arthritis (ERA). METHODS: During a 7-year followup period, all consecutive patients fulfilling the ILAR classification criteria for ERA were enrolled. Data collected included demographic, clinical and laboratory variables at disease onset, at the onset of inflammatory back pain, and at the last available followup visit. Pelvis radiographs and dynamic magnetic resonance imaging (MRI) scans for SI joints were obtained simultaneously in all patients who developed inflammatory back pain. RESULTS: Fifty-nine children with ERA were studied; 40 male, 19 female; median age at disease onset 9 years 4 months (range 6 yrs 6 mo - 13 yrs 3 mo). At a median interval after disease onset of 1 year 3 months, 21 children reported symptoms of inflammatory back pain. In all cases, radiographs of SI joints were negative, while dynamic MRI revealed acute sacroiliitis in 17 cases. Multivariate analysis showed that the early predictors of SI were the number of active joints (p < 0.03) and the number of active entheses (p < 0.001) at onset. CONCLUSION: In our cohort, roughly 30% of children with ERA/juvenile idiopathic arthritis develop clinical and MRI evidence of sacroiliitis, detectable with dynamic MRI as early as 1 year after disease onset. Additional data from larger case series are needed to assess the specificity and sensitivity of this technique in the early phase of the disease and to confirm the rate of SI involvement reported in this cohort.