Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Neuroprotective effects of maternal melatonin administration in early-onset placental insufficiency and fetal growth restriction.

BACKGROUND: Early-onset fetal growth restriction (FGR) is associated with adverse outcomes. We hypothesised that maternal melatonin administration will improve fetal brain structure in FGR. METHODS: Surgery was performed on twin-bearing ewes at 88 days (0.6 gestation), and FGR induced in one twin via single umbilical artery ligation. Melatonin was administered intravenously (6 mg/day) to a group of ewes commencing on day of surgery until 127 days (0.85 gestation), when the ewe/fetuses were euthanized, and fetal brains collected. RESULTS: Study groups were control (n = 5), FGR (n = 5), control+melatonin (control+MLT; n = 6) and FGR+melatonin (FGR + MLT; n = 6). Melatonin administration did not significantly alter fetal body or brain weights. Myelin (CNPase+) fibre density was reduced in FGR vs. control animals in most brain regions examined (p < 0.05) and melatonin treatment restored CNPase fibre density. Similar but less pronounced effect was seen with mature myelin (MBP+) staining. Significant differences in activated microglia (Iba-1) activity were seen between lamb groups (MLT mitigated FGR effect) in periventricular white matter, subventricular zone and external capsule (p < 0.05). Similar effects were seen in astrogliosis (GFAP) in intragyral white matter and cortex. CONCLUSIONS: Maternal melatonin administration in early onset FGR led to improved myelination of white matter brain regions, possibly mediated by decreased inflammation. IMPACT: Maternal melatonin administration might lead to neuroprotection in the growth-restricted fetus, possibly via dampening neuroinflammation and enhancing myelination. This preclinical study adds to the body of work on this topic, and informs clinical translation. Neuroprotection likely to improve long-term outcomes of this vulnerable infant group.

Social support among English learners with disabilities and other adolescents.

INTRODUCTION: Social support is important for many youth but may be particularly important for English learners (ELs) with disabilities, a population that has historically faced barriers accessing resources to meet their educational needs. The current study investigates social support from parents, peers, teachers, and schools in a nationally representative sample of adolescents. METHOD: Data from the National Longitudinal Transition Study 2012 was used to evaluate potential group differences in social support among participants that included ELs with (n = 440) and without disabilities (n = 100) and non-ELs with (n = 4890) and without disabilities (n = 1090). A multivariate analysis of covariance was conducted to evaluate potential between-group variations in social support among these student groups after controlling for variations in background demographic characteristics. RESULTS AND CONCLUSIONS: Results showed between group differences in parental support and peer connectedness but not in teacher or school support. Parents of students with disabilities reported the highest levels of support, whereas parents of ELs without disabilities reported the lowest levels of support. Students with disabilities reported the lowest levels of peer connectedness among the four groups. Overall, levels of teacher and school supports were high across all four groups of students. These patterns contribute to our understanding of the social support network of ELs with disabilities in comparison to other students. Further investigation is needed to examine the mechanisms that contribute to these differences.

The medullary serotonergic centres involved in cardiorespiratory control are disrupted by fetal growth restriction.

Fetal growth restriction (FGR) is associated with cardiovascular and respiratory complications after birth and beyond. Despite research showing a range of neurological changes following FGR, little is known about how FGR affects the brainstem cardiorespiratory control centres. The primary neurons that release serotonin reside in the brainstem cardiorespiratory control centres and may be affected by FGR. At two time points in the last trimester of sheep brain development, 110 and 127 days of gestation (0.74 and 0.86 of gestation), we assessed histopathological alterations in the brainstem cardiorespiratory control centres of the pons and medulla in early-onset FGR versus control fetal sheep. The FGR cohort were hypoxaemic and asymmetrically growth restricted. Compared to the controls, the brainstem of FGR fetuses exhibited signs of neuropathology, including elevated cell death and reduced cell proliferation, grey and white matter deficits, and evidence of oxidative stress and neuroinflammation. FGR brainstem pathology was predominantly observed in the medullary raphé nuclei, hypoglossal nucleus, nucleus ambiguous, solitary tract and nucleus of the solitary tract. The FGR groups showed imbalanced brainstem serotonin and serotonin 1A receptor abundance in the medullary raphé nuclei, despite evidence of increased serotonin staining within vascular regions of placentomes collected from FGR fetuses. Our findings demonstrate both early and adaptive brainstem neuropathology in response to placental insufficiency. KEY POINTS: Early-onset fetal growth restriction (FGR) was induced in fetal sheep, resulting in chronic fetal hypoxaemia. Growth-restricted fetuses exhibit persistent neuropathology in brainstem nuclei, characterised by disrupted cell proliferation and reduced neuronal cell number within critical centres responsible for the regulation of cardiovascular and respiratory functions. Elevated brainstem inflammation and oxidative stress suggest potential mechanisms contributing to the observed neuropathological changes. Both placental and brainstem levels of 5-HT were found to be impaired following FGR.

Cardiovascular decline in offspring during the perinatal period in an ovine model of fetal growth restriction.

Fetal growth restriction (FGR) increases the risk cardiovascular disease (CVD) in adulthood. Placental insufficiency and subsequent chronic fetal hypoxemia are causal factors for FGR, leading to a redistribution of blood flow that prioritizes vital organs. Subclinical signs of cardiovascular dysfunction are evident in growth-restricted neonates; however, the mechanisms programming for CVD in adulthood remain unknown. This study aimed to determine the potential mechanisms underlying structural and functional changes within the heart and essential (carotid) and nonessential (femoral) vascular beds in growth-restricted lambs. Placental insufficiency was surgically induced in ewes at 89 days gestational age (dGA, term = 148dGA). Three age groups were investigated: fetal (126dGA), newborn (24 h after preterm birth), and 4-wk-old lambs. In vivo and histological assessments of cardiovascular indices were undertaken. Resistance femoral artery function was assessed via in vitro wire myography and blockade of key vasoactive pathways including nitric oxide, prostanoids, and endothelium-dependent hyperpolarization. All lambs were normotensive throughout the first 4 wk of life. Overall, the FGR cohort had more globular hearts compared with controls (P = 0.0374). A progressive decline in endothelium-dependent vasodilation was demonstrated in FGR lambs compared with controls. Further investigation revealed that impairment of the prostanoid pathway may drive this reduction in vasodilatory capacity. Clinical indicators of CVD were not observed in our FGR lambs. However, subclinical signs of cardiovascular dysfunction were present in our FGR offspring. This study provides insight into potential mechanisms, such as the prostanoid pathway, that may warrant therapeutic interventions to improve cardiovascular development in growth-restricted newborns.NEW & NOTEWORTHY Our findings provide novel insight into the potential mechanisms that program for cardiovascular dysfunction in growth-restricted neonates as our growth-restricted lambs exhibited a progressive decline in endothelium-dependent vasodilation in the femoral artery between birth and 4 wk of age. Subsequent analyses indicated that this reduction in vasodilatory capacity is likely to be mediated by the prostanoid pathway and prostanoids could be a potential target for therapeutic interventions for fetal growth restriction (FGR).

Ganaxolone versus Phenobarbital for Neonatal Seizure Management.

OBJECTIVE: Seizures are more common in the neonatal period than at any other stage of life. Phenobarbital is the first-line treatment for neonatal seizures and is at best effective in approximately 50% of babies, but may contribute to neuronal injury. Here, we assessed the efficacy of phenobarbital versus the synthetic neurosteroid, ganaxolone, to moderate seizure activity and neuropathology in neonatal lambs exposed to perinatal asphyxia. METHODS: Asphyxia was induced via umbilical cord occlusion in term lambs at birth. Lambs were treated with ganaxolone (5mg/kg/bolus then 5mg/kg/day for 2 days) or phenobarbital (20mg/kg/bolus then 5mg/kg/day for 2 days) at 6 hours. Abnormal brain activity was classified as stereotypic evolving (SE) seizures, epileptiform discharges (EDs), and epileptiform transients (ETs) using continuous amplitude-integrated electroencephalographic recordings. At 48 hours, lambs were euthanized for brain pathology. RESULTS: Asphyxia caused abnormal brain activity, including SE seizures that peaked at 18 to 20 hours, EDs, and ETs, and induced neuronal degeneration and neuroinflammation. Ganaxolone treatment was associated with an 86.4% reduction in the number of seizures compared to the asphyxia group. The total seizure duration in the asphyxia+ganaxolone group was less than the untreated asphyxia group. There was no difference in the number of SE seizures between the asphyxia and asphyxia+phenobarbital groups or duration of SE seizures. Ganaxolone treatment, but not phenobarbital, reduced neuronal degeneration within hippocampal CA1 and CA3 regions, and cortical neurons, and ganaxolone reduced neuroinflammation within the thalamus. INTERPRETATION: Ganaxolone provided better seizure control than phenobarbital in this perinatal asphyxia model and was neuroprotective for the newborn brain, affording a new therapeutic opportunity for treatment of neonatal seizures. ANN NEUROL 2022;92:1066-1079.

An Optimized and Detailed Step-by-Step Protocol for the Analysis of Neuronal Morphology in Golgi-Stained Fetal Sheep Brain.

Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure gray matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1-3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi-staining protocols have been optimized, and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi-stained tissue is then visualized under light microscopy and image analysis may be completed with several software programs for morphological analysis of neurons, including freeware and commercial products. Each program requires optimization, whether semiautomated or automated, requiring different levels of investigator intervention and interpretation, which is a critical consideration for unbiased analysis. Detailed protocols for fetal ovine brain tissue are lacking, and therefore, we provide a step-by-step workflow of computer software analysis for morphometric quantification of Golgi-stained neurons. Here, we utilized the commonly applied FD Rapid GolgiStain kit (FD NeuroTechnologies) on ovine fetal brains collected at 127 days (0.85) of gestational age for the analysis of CA1 pyramidal neurons in the hippocampus. We describe the step-by-step protocol to retrieve neuronal morphometrics using Imaris imaging software to provide quantification of apical and basal dendrites for measures of dendrite length (µm), branch number, branch order, and Sholl analysis (intersections over radius). We also detail software add-ons for data retrieval of dendritic spines including the number of spines, spine density, and spine classification, which are critical indicators of synaptic function. The assessment of neuronal morphology in the developing brain using Rapid-Golgi and Imaris software is labor-intensive, particularly during the optimization period. The methodology described in this step-by-step description is novel, detailed, and aims to provide a reproducible, working protocol to quantify neuronal cytoarchitecture with simple descriptions that will save time for the next users of these commonly used techniques.

Effect of expansion of human umbilical cord blood CD34 + cells on neurotrophic and angiogenic factor expression and function.

The use of CD34 + cell-based therapies has largely been focused on haematological conditions. However, there is increasing evidence that umbilical cord blood (UCB) CD34 + -derived cells have neuroregenerative properties. Due to low cell numbers of CD34 + cells present in UCB, expansion is required to produce sufficient cells for therapeutic purposes, especially in adults or when frequent applications are required. However, it is not known whether expansion of CD34 + cells has an impact on their function and neuroregenerative capacity. We addressed this knowledge gap in this study, via expansion of UCB-derived CD34 + cells using combinations of LDL, UM171 and SR-1 to yield large numbers of cells and then tested their functionality. CD34 + cells expanded for 14 days in media containing UM171 and SR-1 resulted in over 1000-fold expansion. The expanded cells showed an up-regulation of the neurotrophic factor genes BDNF, GDNF, NTF-3 and NTF-4, as well as the angiogenic factors VEGF and ANG. In vitro functionality testing showed that these expanded cells promoted angiogenesis and, in brain glial cells, promoted cell proliferation and reduced production of reactive oxygen species (ROS) during oxidative stress. Collectively, this study showed that our 14-day expansion protocol provided a robust expansion that could produce enough cells for therapeutic purposes. These expanded cells, when tested in in vitro, maintained functionality as demonstrated through promotion of cell proliferation, attenuation of ROS production caused by oxidative stress and promotion of angiogenesis.

Factors affecting healthcare pathways for chronic lung disease management in Vietnam: a qualitative study on patients' perspectives.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) and asthma rank among the leading causes of respiratory morbidity, particularly in low- and middle-income countries. This qualitative study aimed to explore the healthcare pathways of patients with chronic respiratory disease, and factors influencing their ability to access healthcare in Vietnam, where COPD and asthma are prevalent. METHODS: We conducted 41 in-depth interviews among patients, including 31 people with COPD, eight with asthma and two with asthma-COPD overlap syndrome. Participants were recruited at provincial- or national-level health facilities in two urban and two rural provinces in Vietnam. The interviews were audio-recorded, transcribed, and analysed using thematic analysis. RESULTS: Patients' healthcare pathways were complex and involved visits to multiple health facilities before finally obtaining a definitive diagnosis at a provincial- or national-level hospital. Access to healthcare was affected considerably by participants' limited knowledge of their respiratory conditions, the availability of social support, especially from family members, the costs of healthcare as well as health system factors (including the coverage of public health insurance, the distance to health facilities, and attitude of healthcare providers). CONCLUSION: The study demonstrated the need for improved access to timely diagnosis and treatment of chronic lung disease within the lower level of the health system. This can be achieved by enhancing the communication skills and diagnostic capacity of local healthcare workers. Health education programmes for patients and caregivers will contribute to improved control of lung disease.

Evaluating management strategies for sustainable crop production under changing climate conditions: A system dynamics approach.

The increasing frequency and severity of drought pose significant threats to sustainable agricultural production across the world. Managing drought risks is challenging given the complexity of the interdependencies and feedback between climate drivers and socio-economic and ecological systems. To better understand the dynamics that drive the impacts of drought and water scarcity on crop production, a system dynamics model has been developed to explore complex interactions between factors in associated with drought and agricultural production, and examine how these might impact agricultural sustainability, using a case study in a coffee production system in Viet Nam. The model shows that water- and land-use drivers and their interactions with ecological and socio-economic factors play a more significant role than drought in determining the sustainability of coffee production. Results of policy scenario analyses indicate that drought conditions might exacerbate problems related to water shortages for irrigation but their impacts could be substantially minimized through applying intervention strategies, including restriction of the total area of land available for coffee production (to ~ 190,000 ha) and a 25% reduction in the irrigation amount per hectare of coffee compared to the common practices. Overall, the model findings add significant insight into drought and water resources management for sustainable crop production and the developed model can serve as a decision-support tool to inform strategic policy-making.

Maternal ABVD chemotherapy for Hodgkin lymphoma in a dichorionic diamniotic pregnancy: a case report.

BACKGROUND: Hodgkin lymphoma (HL) is the most common hematological malignancy during pregnancy. The first-line treatment for HL in pregnancy is the standard ABVD regimen without any drug and/or dose adjustment. However, data on chemotherapy during twin pregnancies are sparse, and a better understanding of the mechanisms involved in exposure to and the toxic effects of anticancer drugs in the fetuses is needed. CASE PRESENTATION: A 41-year-old dichorionic diamniotic pregnant patient was given ABVD treatment for HL at a gestational age of 28 weeks and 3 days. The patient received 2 cycles of chemotherapy with a 15-day therapeutic window including an actual 25 mg/m2 dose of doxorubicin per cycle. Unlike the female twin, the male twin presented four days after birth a left cardiac dysfunction. Doxorubicin cardiotoxicity in the male newborn was also supported by high blood levels of troponin. At one month of age, echocardiography findings were normal. We investigated literature data on physiological aspects of pregnancy that may influence doxorubicin pharmacokinetics, and pharmacodynamic and pharmacokinetic data on the use of doxorubicin in pregnancy. We detailed the role of the transporters in doxorubicin placenta distribution, and tried to understand why only one fetus was affected. CONCLUSIONS: Fetal safety depends at least on maternal doxorubicin pharmacokinetics.Because of drug interactions (i.e. drug metabolism and drug transport), care should always be taken to avoid maternal pharmacokinetic variability. The toxic effects were discrepant between the dizygotic twins, suggesting additional fetus-specific pharmacokinetic/pharmacodynamic factors in doxorubicin toxicity.

Umbilical cord blood versus mesenchymal stem cells for inflammation-induced preterm brain injury in fetal sheep.

BACKGROUND: Chorioamnionitis and fetal inflammation are principal causes of neuropathology detected after birth, particularly in very preterm infants. Preclinical studies show that umbilical cord blood (UCB) cells are neuroprotective, but it is uncertain if allogeneic UCB cells are a feasible early intervention for preterm infants. In contrast, mesenchymal stem cells (MSCs) are more readily accessible and show strong anti-inflammatory benefits. We aimed to compare the neuroprotective benefits of UCB versus MSCs in a large animal model of inflammation-induced preterm brain injury. We hypothesized that MSCs would afford greater neuroprotection. METHODS: Chronically instrumented fetal sheep at 0.65 gestation received intravenous lipopolysaccharide (150 ng; 055:B5, n = 8) over 3 consecutive days; or saline for controls (n = 8). Cell-treated animals received 108 UCB mononuclear cells (n = 7) or 107 umbilical cord MSCs (n = 8), intravenously, 6 h after the final lipopolysaccharide dose. Seven days later, cerebrospinal fluid and brain tissue was collected for analysis. RESULTS: Lipopolysaccharide induced neuroinflammation and apoptosis, and reduced the number of mature oligodendrocytes. MSCs reduced astrogliosis, but UCB did not have the same effect. UCB significantly decreased cerebral apoptosis and protected mature myelinating oligodendrocytes, but MSCs did not. CONCLUSION: UCB appears to better protect white matter development in the preterm brain in response to inflammation-induced brain injury in fetal sheep.

Neuropathology as a consequence of neonatal ventilation in premature growth-restricted lambs.

Fetal growth restriction (FGR) and prematurity are associated with high risk of brain injury and long-term neurological deficits. FGR infants born preterm are commonly exposed to mechanical ventilation, but it is not known whether ventilation differentially induces brain pathology in FGR infants compared with appropriate for gestational age (AGA) infants. We investigated markers of neuropathology in moderate- to late-preterm FGR lambs, compared with AGA lambs, delivered by caesarean birth and ventilated under standard neonatal conditions for 24 h. FGR was induced by single umbilical artery ligation in fetal sheep at 88-day gestation (term, 150 days). At 125-day gestation, FGR and AGA lambs were delivered, dried, intubated, and commenced on noninjurious ventilation, with surfactant administration at 10 min. A group of unventilated FGR and AGA lambs at the same gestation was also examined. Over 24 h, circulating pH, Po2, and lactate levels were similar between groups. Ventilated FGR lambs had lower cerebral blood flow compared with AGA lambs ( P = 0.01). The brain of ventilated FGR lambs showed neuropathology compared with unventilated FGR, and unventilated and ventilated AGA lambs, with increased apoptosis (caspase-3), blood-brain barrier dysfunction (albumin extravasation), activated microglia (Iba-1), and increased expression of cellular oxidative stress (4-hydroxynonenal). The neuropathologies seen in the ventilated FGR brain were most pronounced in the periventricular and subcortical white matter but also evident in the subventricular zone, cortical gray matter, and hippocampus. Ventilation of preterm FGR lambs increased brain injury compared with AGA preterm lambs and unventilated FGR lambs, mediated via increased vascular permeability, neuroinflammation and oxidative stress.

Immunosuppression in pediatric liver transplant recipients: Unique aspects.

Pediatric liver transplantation has experienced improved outcomes over the last 50 years. This can be attributed in part to establishing optimal use of immunosuppressive agents to achieve a balance between minimizing the risks of allograft rejection and infection. The management of immunosuppression in children is generally more complex and can be challenging when compared with the use of these agents in adult liver transplant patients. Physiologic differences in children alter the pharmacokinetics of immunosuppressive agents, which affects absorption, distribution, metabolism, and drug excretion. Children also have a longer expected period of exposure to immunosuppression, which can impact growth, risk of infection (bacterial, viral, and fungal), carcinogenesis, and likelihood of nonadherence. This review discusses immunosuppressive options for pediatric liver transplant recipients and the unique issues that must be addressed when managing this population. Further advances in the field of tolerance and accommodation are needed to relieve the acute and cumulative burden of chronic immunosuppression in children. Liver Transplantation 23 244-256 2017 AASLD.

Cord blood mononuclear cells prevent neuronal apoptosis in response to perinatal asphyxia in the newborn lamb.

Perinatal asphyxia is a significant cause of death or long-term neurodevelopmental impairment. Hypothermia, currently the only effective treatment, leads to modest improvements, but new therapeutic strategies are required. Umbilical cord blood (UCB) mononuclear cells have potent anti-inflammatory properties and may reduce neuropathology. This study examined whether autologous UCB mononuclear cells were neuroprotective when administered to newborn lambs at 12 h after birth asphyxia. At caesarean section, birth asphyxia was induced by clamping the umbilical cord until mean arterial blood pressure decreased to 18-20 mmHg. Asphyxia (n = 20) or control (n = 11) lambs were resuscitated and maintained, with magnetic resonance spectroscropy (MRS) performed at 12 and 72 h, and were then killed at 72 h. Cord blood was collected once the cord was clamped, and mononuclear cells were isolated and labelled fluorescently and administered to control (n = 3) or asphyxia (n = 8) lambs. Asphyxia induced a significant increase in cellular apoptosis (caspase-3 immunopositive) within all brain regions examined, including cortex, hippocampus, thalamus, striatum and subcortical white matter (P < 0.01 vs. control). Additionally, asphyxia induced significant and widespread astrogliosis and increased inflammatory cells (activated microglia and macrophages). The administration of UCB mononuclear cells (asphyxia+UCB) significantly decreased neuronal apoptosis, astrogliosis and inflammation (P < 0.05 vs. asphyxia alone). Asphyxia+UCB lambs also demonstrated decreased brain metabolites lactate:choline (P = 0.01) and lactate:N-acetylaspartate (P < 0.01) from 12 to 72 h, detected using MRS. Autologous UCB mononuclear cell treatment restores normal brain metabolism following perinatal asphyxia, and reduces brain inflammation, astrogliosis and neuronal apoptosis, supporting its use as a neuroprotective therapy following asphyxia.

Prevalence of Pancreatic Steatosis at a Pediatric Tertiary Care Center.

OBJECTIVES: Pancreatic steatosis in adults has been proposed to be associated with obesity; however, data on pancreatic steatosis in children are lacking. Our study aimed to measure the prevalence of pancreatic steatosis in children and to examine its association with obesity and nonalcoholic fatty liver disease. METHODS: This is a retrospective chart review study of 232 patients 2 to 18 years old who underwent abdominal computed tomographic imaging in the emergency department or inpatient ward within a 1-year time span and from whom demographics, anthropometrics, and medical history were obtained. Our radiologist determined mean Hounsfield unit (HU) measurements for the pancreas, liver, and spleen. A difference of -20 between the pancreas and spleen (psHU) and between the liver and spleen was used to determine fatty infiltration. RESULTS: Of the 232 patients, 11.5% had a psHU less than -20. The prevalence of pancreatic steatosis was more than double among obese children (19%) than that in nonobese groups (8%). There is a significant correlation between the psHU and liver-spleen HU (r = 0.50, P < 0.001). CONCLUSIONS: Pancreatic steatosis was identified in 10% of the study population and is associated with obesity. Also, pancreatic steatosis is significantly associated with nonalcoholic fatty liver disease. This is the first study assessing the prevalence of pancreatic steatosis in children.

Evaluation of the safety and tolerability of a high-dose intravenous infusion of allogeneic mesenchymal precursor cells.

BACKGROUND AIMS: Over the past decade, mounting evidence has shown that mesenchymal stromal cells have the potential to exert protective and reparative effects in a variety of disease settings. Clinical trials are being increasingly established to investigate the therapeutic potential of these cells; however, several safety concerns remain to be addressed, of which dosage safety for intravenous administration is paramount. Published safety studies thus far have predominantly been carried out in small-animal models, whereas data for high-dose allogeneic intravenous administration in large-animal models are limited. This study investigates the safety and tolerability of a single high-dose intravenous infusion of 450 million allogeneic ovine mesenchymal precursor cells (oMPCs) in adult sheep. METHODS: Allogeneic oMPCs (n = 450 million) were intravenously administered to 2-year-old castrated male sheep through the use of three different infusion rates. Sheep were intensively monitored for 7 days by means of vital physiological observations (temperature, blood pressure, heart rate, respiratory rate and oxygen saturation) as well as venous and arterial blood analysis. In addition, full post mortem examination was performed in all animals. RESULTS: A single high dose of intravenously administered cells was well tolerated, with no serious adverse effects reported. No physiologically significant changes in vital signs, oxygen saturation, blood gas analysis or clinical pathology were observed over the duration of the study. CONCLUSIONS: Intravenous delivery of a single high-dose infusion of oMPCs is well tolerated in a large animal model. This study provides additional safety evidence for their intravenous use in future human clinical trials.

An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application.

Measuring help-seeking behaviors: factor structure, reliability, and validity among youth with disabilities.

Existing measures of help-seeking focus on assessing attitudes and beliefs, rather than specific behaviors, toward help-seeking. This study described the development of a self-report measure of informal help-seeking behaviors (HSB). Participants were 228 high school students (148 males, 80 females) with disabilities from four states. Factor analyses revealed three underlying factors, each addressing a different source of help: parent, peer, and teacher. The HSB had good internal reliability and moderate validity. Results from regression analyses suggested that help-seeking behaviors toward parents and teachers contributed uniquely to students' self-ratings of school bonding, life satisfaction, and career outcome expectations. Help-seeking behaviors toward peers was a negative predictor of career outcome expectations. The value of the HSB as a research instrument was discussed.

Immunosuppressive Cyclotides: A Promising Approach for Treating Autoimmune Diseases.

The immune system maintains constant surveillance to prevent the infiltration of both endogenous and exogenous threats into host organisms. The process is regulated by effector immune cells that combat external pathogens and regulatory immune cells that inhibit excessive internal body inflammation, ultimately establishing a state of homeostasis within the body. Disruption to this process could lead to autoimmunity, which is often associated with the malfunction of both T cells and B cells with T cells playing a more major role. A number of therapeutic mediators for autoimmune diseases are available, from conventional disease-modifying drugs to biologic agents and small molecule inhibitors. Recently, ribosomally synthesized peptides, specifically cyclotides from plants are currently attracting more attention as potential autoimmune disease therapeutics due to their decreased toxicity compared to small molecules inhibitors as well as their remarkable stability against a number of factors. This review provides a concise overview of various cyclotides exhibiting immunomodulatory properties and their potential as therapeutic interventions for autoimmune diseases.