Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (IT) versus intravenous (IV) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the IT (5×10⁵) or IV (2×10⁶) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both IT and IV transplantations. However, IT administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to IV administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the IT group, but not in the IV group. Although the IT group received only one fourth of the number of MSCs that the IV group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the IT group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the IT group, but not in the IV group. Thus, local IT MSC transplantation was more effective than systemic IV MSC administration in protecting against neonatal hyperoxic lung injury.     

Repeat Diagnoses of Bethesda Category III Thyroid Nodules: What To Do Next?

Objectives

To assess the malignancy rates of thyroid nodules repeatedly classified as Bethesda category III on fine needle aspiration (FNA), and to suggest management guidelines for these lesions.

Methods

This is a retrospective study that included 395 thyroid nodules categorized as Bethesda III undergone either surgery or ultrasound (US) follow-up. There were 67 nodules classified a second time as Bethesda category III on repeat FNA. We compared malignancy rates, clinicopathologic and ultrasonographic characteristics between direct surgery and repeat FNA groups and between the initial and repeat Bethesda category III groups, each. And in the repeat Bethesda III group, clinicopathologic and US variables were compared between benign and malignant nodules.

Results

Incidence of concurrent cancer, underlying thyroiditis and positive BRAF mutation were significantly higher in 142 nodules with direct surgery than 243 nodules with repeat FNA (p < 0.05). Of the 395 nodules with Bethesda category III cytology on initial FNA, the malignancy rate was 59.5%. In 67 nodules with repeat Bethesda III classification, however, the malignancy rate was 73.1% (p < 0.05). However, none of the variables were significantly different between the initial Bethesda category III group and the repeat Bethesda category III group (p > 0.05). In the repeat Bethesda category III group, solid consistency, irregular/microlobulated margins, nonparallel shape, and number of suspicious findings or “suspicious malignant” US assessments were associated with a high malignancy rate (p < 0.05). On multivariate logistic regression analysis, the factor associated with malignancy in the repeat Bethesda category III group was irregular/microlobulated margin (odds ratio = 15.576; 95% CI, 2.097–115.6804, p = 0.007) with a sensitivity, specificity, positive and negative predictive values, and accuracy of 81.6%, 83.3%, 93.0%, 62.5% and 82.1%, respectively.

Conclusion

Thyroid nodules with repeated Bethesda category III classification and irregular/microlobulated margins on US are at increased risk of malignancy, and operative management should be considered as opposed to repeat FNA.     

Modeling of Menkes disease via human induced pluripotent stem cells

Menkes disease (MD) is a copper-deficient neurodegenerative disorder that manifests severe neurologic symptoms such as seizures, lethargic states, and hypotonia. Menkes disease is due to a dysfunction of ATP7A, but the pathophysiology of neurologic manifestation is poorly understood during embryonic development. To understand the pathophysiology of neurologic symptoms, molecular and cellular phenotypes were investigated in Menkes disease-derived induced pluripotent stem cells (MD-iPSCs). MD-iPSCs were generated from fibroblasts of a Menkes disease patient. Abnormal reticular distribution of ATP7A was observed in MD-fibroblasts and MD-iPSCs, respectively. MD-iPSCs showed abnormal morphology in appearance during embryoid body (EB) formation as compared with wild type (WT)-iPSCs. Intriguingly, aberrant switch of E-cadherin (E-cad) to N-cadherin (N-cad) and impaired neural rosette formation were shown in MD-iPSCs during early differentiation. When extracellular copper was chelated in WT-iPSCs by treatment with bathocuprione sulfate, aberrant switch of E-cad to N-cad and impaired neuronal differentiation were observed, like in MD-iPSCs. Our results suggest that neurological defects in Menkes disease patients may be responsible for aberrant cadherin transition and impaired neuronal differentiation during early developmental stage.     

Increased centrosome amplification in aged stem cells of the Drosophila midgut

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.     

MT2 Melatonin Receptor Immunoreactivity in Neurons is Very High in the Aged Hippocampal Formation in Gerbils

Melatonin exerts many physiological functions via its G protein-coupled receptors. In the present study, we investigated age-related changes in MT2 melatonin receptor immunoreactivity and its levels in the gerbil hippocampus during normal aging. In the postnatal month 1 (PM 1) group, MT2 immunoreaction was well observed in neurons in all subregions of the gerbil hippocampus. In the PM 3 and 6 groups, MT2 immunoreactivity in neurons was decreased compared to that in the PM 1 group. Thereafter, MT2 immunoreactivity in neurons was increased. In the PM 18 and 24 groups, MT2 immunoreactivity in neurons was strong in all subregions of the gerbil hippocampus. In addition, the number of MT2 immunoreactive cells was lowest at PM 3 and highest at PM 24. From western blot analysis, age-dependent change pattern in MT2 level in the gerbil hippocampus was similar to the immunohistochemical result. These results indicate that MT2 immunoreactivity and levels are altered in the gerbil hippocampus during normal aging; lowest at young adult stage and highest at aged stage.     

Systemic Administration of Lipopolysaccharide Induces Cyclooxygenase-2 Immunoreactivity in Endothelium and Increases Microglia in the Mouse Hippocampus

In this study, we observed the effects of lipopolysaccharide (LPS) on neurodegeneration and immune response in the hippocampus. LPS is a gram-negative bacterial cell surface proteoglycan and known as a bacterial endotoxin. For this, we investigated the optimal concentration of LPS influencing the ICR mouse hippocampus to measure the LPS receptor, e.g., toll-like receptor 4 (TLR4), expression in mouse hippocampal homogenates. TLR4 expression was significantly and prominently increased in the hippocampal homogenates of the LPS (1 mg/kg)-treated group. Next, we examined pro-inflammatory response in the hippocampus using cyclooxygenase-2 (COX-2, a marker for inflammatory response) immunohistochemistry after LPS treatment. COX-2 immunoreactivity was significantly increased in the endothelium of blood vessels in the hippocampus 6 h after LPS treatment, judging from double immunofluorescence study with platelet-derived endothelial cell adhesion molecule-1 (PECAM-1, a marker for endothelial cells): it decreased 12 h and disappeared 24 h after LPS treatment. In addition, the ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive (⁺) microglia were morphologically activated in the mouse hippocampus after LPS treatment. At 24 h after LPS treatment, Iba-1⁺ microglia of activated forms were abundant in the hippocampus. However, NeuN (a neuron-specific soluble nuclear antigen)⁺ neurons were not significantly changed in the hippocampus after LPS treatment. Fluoro-jade B (a marker for neuronal degeneration)⁺ cells were not detected in the hippocampus at any time after LPS treatment. In addition, there were no significant differences in permeability of blood–brain barriers at any time points after LPS treatment. In brief, our results indicate that intraperitoneal administration of 1 mg/kg LPS effectively induces LPS receptor (TLR4) expression in the hippocampus, and the treatment increases corticosterone levels, inflammation in the blood vessels, and microglial activation in the hippocampus without any neuronal damage.     

Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

The inositol 1,4,5-trisphosphate (IP₃)-mediated intracellular Ca²⁺ releases in secretory cells play vital roles in controlling not only the intracellular Ca²⁺ concentrations but also the Ca²⁺-dependent exocytotic processes. Of intracellular organelles that release Ca²⁺ in response to IP₃, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP₃-dependent Ca²⁺ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP₃-mediated Ca²⁺ release as well as the presence of the IP₃ receptor (IP₃R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP₃R isoforms, and 58–69% of total cellular IP₃R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin II are high-capacity, low-affinity Ca²⁺ binding proteins, binding 30–93 mol of Ca²⁺/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca²⁺ storage properties of chromogranins secretory granules contain ~40 mM Ca²⁺. Furthermore, chromogranins A and B directly interact with the IP₃Rs and modulate the IP₃R/Ca²⁺ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively. Coupled chromogranins change the IP₃R/Ca²⁺ channels to a more ordered, release-ready state, whereby making the IP₃R/Ca²⁺ channels significantly more sensitive to IP₃.     

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity and its protein levels in the gerbil hippocampus during normal aging

Phosphorylated extracellular signal-regulated kinase (pERK) mediates neuronal synaptic plasticity, long-term potentiation, and learning and memory in the hippocampus. In this study, we examined pERK1/2 immunoreactivity and its protein level in the gerbil hippocampus at various ages. In the postnatal month 1 (PM 1) group, very weak pERK1/2 immunoreactivity was detected in the hippocampus. In the CA1 region, pERK1/2 immunoreactivity was considerably increased in the stratum pyramidale in the PM 6 group. Thereafter, pERK1/2 immunoreactivity was decreased. In the CA2/3 region, pERK1/2 immunoreactivity increased in an age-dependent manner until PM 12. Thereafter, numbers of pERK1/2-immunoreactive neurons were decreased. However, in the mossy fiber zone, pERK1/2 immunostaining became stronger with age. In the dentate gyrus, a few pERK1/2-immunoreactive cells were observed until PM 12. In the PM 18 and 24 groups, numbers of pERK1/2-immunoreactive cells were increased, especially in the polymorphic layer. In Western blot analysis, pERK1/2 level in the gerbil hippocampus was increased with age. These results indicate that total pERK1/2 levels are increased in the hippocampus with age. However pERK1/2 immunoreactivity in subregions of the gerbil hippocampus was changed with different pattern during normal aging.     

Development of an amphiphilic matrix for immobilization of <Emphasis Type="Italic">Candida antartica</Emphasis> lipase B for biodiesel production

Biodiesel has been greatly interested as an alternative fuel and is produced by a transesterification reaction of oil with alcohol. Recently, microbial lipases have been used for biodiesel production. Among the microbial lipase, immobilized Candida antartica lipase B (CALB) is the most widely used. However, CALB is unstable and shows low catalytic efficiency in the reaction media because the reaction media contains a high concentration of methanol and the lipase is also inhibited by the by-product glycerol. In this study, to overcome these limitations, we developed an amphiphilic matrix to immobilize CALB. The immobilized lipase in an amphiphilic matrix with 80% ethyltrimethoxysilane (ETMS) in tetramethoxysilane (TMOS) and pretreated with oil showed the highest specific activity and biodiesel conversion ratio; about 90% biodiesel conversion in 24 h at an initial molar ratio of 1: 1 (oil: methanol) with stepwise methanol feeding in order to adjust the net molar ratio to be 1: 3.