Histology and chronological magnetic resonance images of congenital spinal deformity: An experimental study in mice model.

BACKGROUND: The natural history of the congenital spinal deformity and its clinical magnitude vary widely in human species. However, we previously reported that the spinal deformities of congenital scoliosis mice did not progress throughout our observational period according to soft X-ray and MRI data. In this study, congenital vertebral and intervertebral malformations in mice were assessed via magnetic resonance (MR) and histological images. METHODS: Congenital spinal anomalies were chronologically assessed via soft X-ray and 7 T MR imaging. MR images were compared to the histological images to validate the findings around the malformations. RESULTS: Soft X-ray images showed the gross alignment of the spine and the contour of the malformed vertebrae, with the growth plate and cortical bone visible as higher density lines, but could not be used to distinguish the existence of intervertebral structures. In contrast, MR images could be used to distinguish each structure, including the cortical bone, growth plate, cartilaginous end plate, and nucleus pulposus, by combining the signal changes on T1-weighted imaging (T1WI) and T2-weighted imaging (T2WI). The intervertebral structure adjacent to the malformed vertebrae also exhibited various abnormalities, such as growth plate and cartilaginous end plate irregularities, nucleus pulposus defects, and bone marrow formation. In the chronological observation, the thickness and shape of the malformed structures on T1WI did not change. CONCLUSIONS: Spinal malformations in mice were chronologically observed via 7 T MRI and histology. MR images could be used to distinguish the histological structures of normal and malformed mouse spines. Malformed vertebrae were accompanied by adjacent intervertebral structures that corresponded to the fully segmented structures observed in human congenital scoliosis, but the intervertebral conditions varied. This study suggested the importance of MRI and histological examinations of human congenital scoliosis patients with patterns other than nonsegmenting patterns, which may be used to predict the prognosis of patients with spinal deformities associated with malformed vertebrae.

REST Inactivation and Coexpression of ASCL1 and POU3F4 Are Necessary for the Complete Transformation of RB1/TP53-Inactivated Lung Adenocarcinoma into Neuroendocrine Carcinoma.

Although recent reports have revealed the importance of the inactivation of both RB1 and TP53 in the transformation from lung adenocarcinoma into neuroendocrine carcinoma (NEC), the requirements for complete transformation into NEC have not been elucidated. To investigate alterations in the characteristics associated with the inactivation of RB1/TP53 and define the requirements for transformation into NEC cells, RB1/TP53 double-knockout A549 lung adenocarcinoma cells were established, and additional knockout of REST and transfection of ASCL1 and POU class 3 homeobox transcription factors (TFs) was conducted. More than 60 genes that are abundantly expressed in neural cells and several genes associated with epithelial-to-mesenchymal transition were up-regulated in RB1/TP53 double-knockout A549 cells. Although the expression of chromogranin A and synaptophysin was induced by additional knockout of REST (which mimics the status of most NECs), the expression of another neuroendocrine marker, CD56, and proneural TFs was not induced. However, coexpression of ASCL1 and POU3F4 in RB1/TP53/REST triple-knockout A549 cells induced the expression of not only CD56 but also other proneural TFs (NEUROD1 and insulinoma-associated 1) and induced NEC-like morphology. These findings suggest that the inactivation of RB1 and TP53 induces a state necessary for the transformation of lung adenocarcinoma into NEC and that further inactivation of REST and coexpression of ASCL1 and POU3F4 are the triggers for complete transformation into NEC.

Enhancement of glycolysis by inhibition of oxygen-sensing prolyl hydroxylases protects alveolar epithelial cells from acute lung injury.

Cellular bioenergetic failure caused by mitochondrial dysfunction is a key process of alveolar epithelial injury during acute respiratory distress syndrome (ARDS). Prolyl hydroxylases (PHDs) act as cellular oxygen sensors, and their inhibition activates hypoxia-inducible factor (HIF), resulting in enhanced cellular glycolytic activity, which could compensate for impaired mitochondrial function and protect alveolar epithelial cells from ARDS. Here, we evaluated the effects of pharmacological PHD inhibition with dimethyloxalylglycine (DMOG) on alveolar epithelial cell injury using in vitro and in vivo ARDS models. We established an in vitro model of alveolar epithelial injury mimicking ARDS by adding isolated neutrophils and LPS to cultured MLE12 alveolar epithelial cells. DMOG treatment protected MLE12 cells from neutrophil-LPS-induced ATP decline and cell death. Knockdown of HIF-1α or inhibition of glycolysis abolished the protective effect of DMOG, suggesting that it was exerted by HIF-1-dependent enhancement of glycolysis. Additionally, intratracheal DMOG administration to mice protected the alveolar epithelial barrier and improved arterial oxygenation, preventing ATP decline during LPS-induced lung injury. In summary, enhancement of glycolysis by PHD inhibition is a potential therapeutic approach for ARDS, protecting alveolar epithelial cells from bioenergetic failure and cell death.- Tojo, K., Tamada, N., Nagamine, Y., Yazawa, T., Ota, S., Goto, T. Enhancement of glycolysis by inhibition of oxygen-sensing prolyl hydroxylases protects alveolar epithelial cells from acute lung injury. FASEB J. 32, 2258-2268 (2018). www.fasebj.org.

Driving-pressure-independent protective effects of open lung approach against experimental acute respiratory distress syndrome.

BACKGROUND: The open lung approach (OLA) reportedly has lung-protective effects against acute respiratory distress syndrome (ARDS). Recently, lowering of the driving pressure (ΔP), rather than improvement in lung aeration per se, has come to be considered as the primary lung-protective mechanism of OLA. However, the driving pressure-independent protective effects of OLA have never been evaluated in experimental studies. We here evaluated whether OLA shows protective effects against experimental ARDS even when the ΔP is not lowered. METHODS: Lipopolysaccharide was intratracheally administered to rats to establish experimental ARDS. After 24 h, rats were mechanically ventilated and randomly allocated to the OLA or control group. In the OLA group, 5 cmH2O positive end-expiratory pressure (PEEP) and recruitment maneuver (RM) were applied. Neither PEEP nor RM was applied to the rats in the control group. Dynamic ΔP was kept at 15 cmH2O in both groups. After 6 h of mechanical ventilation, rats in both groups received RM to inflate reversible atelectasis of the lungs. Arterial blood gas analysis, lung computed tomography, histological evaluation, and comprehensive biochemical analysis were performed. RESULTS: OLA significantly improved lung aeration, arterial oxygenation, and gas exchange. Even after RM in both groups, the differences in these parameters between the two groups persisted, indicating that the atelectasis-induced respiratory dysfunction observed in the control group is not an easily reversible functional problem. Lung histological damage was severe in the dorsal dependent area in both groups, but was attenuated by OLA. White blood cell counts, protein concentrations, and tissue injury markers in the broncho-alveolar lavage fluid (BALF) were higher in the control than in the OLA group. Furthermore, levels of CXCL-7, a platelet-derived chemokine, were higher in the BALF from the control group, indicating that OLA protects the lungs by suppressing platelet activation. CONCLUSIONS: OLA shows protective effects against experimental ARDS, even when the ΔP is not decreased. In addition to reducing ΔP, maintaining lung aeration seems to be important for lung protection in ARDS.

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 109 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed.

Inhibition of Prolyl Hydroxylase Attenuates Fas Ligand-Induced Apoptosis and Lung Injury in Mice.

Alveolar epithelial injury and increased alveolar permeability are hallmarks of acute respiratory distress syndrome. Apoptosis of lung epithelial cells via the Fas/Fas ligand (FasL) pathway plays a critical role in alveolar epithelial injury. Activation of hypoxia-inducible factor (HIF)-1 by inhibition of prolyl hydroxylase domain proteins (PHDs) is a possible therapeutic approach to attenuate apoptosis and organ injury. Here, we investigated whether treatment with dimethyloxalylglycine (DMOG), an inhibitor of PHDs, could attenuate Fas/FasL-dependent apoptosis in lung epithelial cells and lung injury. DMOG increased HIF-1α protein expression in vitro in MLE-12 cells, a murine alveolar epithelial cell line. Treatment of MLE-12 cells with DMOG significantly suppressed cell surface expression of Fas and attenuated FasL-induced caspase-3 activation and apoptotic cell death. Inhibition of the HIF-1 pathway by echinomycin or small interfering RNA transfection abolished these antiapoptotic effects of DMOG. Moreover, intraperitoneal injection of DMOG in mice increased HIF-1α expression and decreased Fas expression in lung tissues. DMOG treatment significantly attenuated caspase-3 activation, apoptotic cell death in lung tissue, and the increase in alveolar permeability in mice instilled intratracheally with FasL. In addition, inflammatory responses and histopathological changes were also significantly attenuated by DMOG treatment. In conclusion, inhibition of PHDs protects lung epithelial cells from Fas/FasL-dependent apoptosis through HIF-1 activation and attenuates lung injury in mice.

CD8-positive T-cell lymphoproliferative disorder associated with Epstein-Barr virus-infected B-cells in a rheumatoid arthritis patient under methotrexate treatment.

We report a 48-year-old female who developed lymphoproliferative disorder (LPD) during treatment of rheumatoid arthritis (RA) with methotrexate (MTX). She presented with multiple tumors in the cervical lymph nodes (LNs), multiple lung shadows and round shadows in both kidneys with pancytopenia and a high CRP level. The LN showed CD8-positive T-cell LPD associated with Epstein-Barr (EB) virus-infected B-cells. Clonality assays for immunoglobulin (Ig) heavy chain and T-cell receptor gamma (TCRγ) were negative. The cessation of MTX without chemotherapy resulted in the complete disappearance of the tumors and abnormal clinical features. We compared this case with previously published ones and discuss the pathological findings, presuming that the proliferation of CD8 T-cells was a reactive manifestation to reactivated EB virus-infected B-cells.

High-grade fetal adenocarcinoma of the lung is a tumour with a fetal phenotype that shows diverse differentiation, including high-grade neuroendocrine carcinoma: a clinicopathological, immunohistochemical and mutational study of 20 cases.

AIMS: High-grade fetal adenocarcinoma (H-FLAC) is a rare variant of pulmonary adenocarcinoma; this study aims to elucidate its clinicopathological features and genetic abnormalities. METHODS AND RESULTS: Clinicopathological, immunohistochemical and mutational analyses were performed on 20 surgically resected lung cancers that showed H-FLAC histology in various proportions. These tumours predominantly occurred in elderly males and in 10 patients who were heavy smokers. Four cases were pure H-FLAC, and 16 cases were mixed H-FLAC, which were found to be combined with conventional-type adenocarcinoma (15 cases), large-cell neuroendocrine carcinoma (three cases), small-cell carcinoma (one case), enteric adenocarcinoma (two cases), choriocarcinoma (two cases), and a solid-clear cell pattern (seven cases). The fetal phenotype and diverse differentiation were supported by the immunoexpression of α-fetoprotein (95%), thyroid transcription factor-1 (TTF-1) (50%), neuroendocrine markers (30-45%), proneural markers (50-69%), and CDX2 (40%). Except for TTF-1 expression (pure H-FLACs, 0%; mixed H-FLACs, 63%), there were no significant differences in histological or immunohistochemical findings between pure and mixed H-FLACs. EGFR, KRAS, BRAF and PIK3CA mutations were identified in 20%, 0%, 0% and 7% of the tumours, respectively. CONCLUSIONS: Lung adenocarcinomas with H-FLAC features possess the potential for multidirectional differentiation, and are not strongly associated with known major driver gene mutations.

Edaravone prevents lung injury induced by hepatic ischemia-reperfusion.

BACKGROUND: Lung injury is a major clinical concern after hepatic ischemia-reperfusion (I/R), due to the production of reactive oxygen species in the reperfused liver. We investigated the efficacy of edaravone, a potent free-radical scavenger, for attenuating lung injury after hepatic I/R. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were assigned to sham + normal saline (NS), I/R + NS, or I/R + edaravone group. Rats in the I/R groups were subjected to 90 min of partial hepatic I/R. Five minutes before reperfusion, 3 mg/kg edaravone was administered to the I/R + edaravone group. After 6 h of reperfusion, we evaluated lung histopathology and wet-to-dry ratio. We also measured malondialdehyde (MDA), an indicator of oxidative stress, in the liver and the lung, as well as cytokine messenger RNA expressions in the reperfused liver and plasma cytokine concentrations. RESULTS: Histopathology revealed lung damages after 6 h reperfusion of partial ischemic liver. Moreover, a significant increase in lung wet-to-dry ratio was observed. MDA concentration increased in the reperfused liver, but not in the lungs. Edaravone administration attenuated the lung injury and the increase of MDA in the reperfused liver. Edaravone also suppressed the reperfusion-induced increase of interleukin-6 messenger RNA expressions in the liver and plasma interleukin-6 concentrations. CONCLUSIONS: Edaravone administration before reperfusion of the ischemic liver attenuates oxidative stress in the reperfused liver and the subsequent lung injury. Edaravone may be beneficial for preventing lung injury induced by hepatic I/R.

Recent advances in histogenesis research of lung neuroendocrine cancers: Evidence obtained from functional analyses of primitive neural/neuroendocrine cell-specific transcription factors.

Small cell carcinoma (SmCC) and large cell neuroendocrine carcinoma (LENEC) are categorized as neuroendocrine cancers (NECs) of the lung and have extremely poor prognoses. The lack of an effective therapeutic strategy against SmCC and LCNEC is a serious issue. Because the regulation of the cellular phenotype is complicated by the actions of various transcription factors, investigations into the function of neural/neuroendocrine cell-specific transcription factors are important for elucidating the cellular characteristics and histogenesis of SmCC and LCNEC and for establishing innovative therapeutic strategies against them. In this review, the functions of ASCL1, NeuroD1, REST, TTF1, and class III/IV POU, that are specifically and highly expressed in lung NECs, are introduced. These transcription factors transactivate and/or transrepress various genes and are involved in neural progenitor phenotyping, neuroendocrine and stem cell marker expression, and epithelial-to-mesenchymal transition. Based on the evidence that certain carcinoids express ASCL1, NeuroD1, TTF1, and class III/IV POU and that lung NECs can develop from non-NE cells/non-NEC cells, the relationships among lung NECs, carcinoid tumors, and non-NECs are discussed. Finally, a model of the histogenesis of lung NECs in view of similarities in the expression of primitive neural/neuroendocrine cell-specific transcription factors is proposed.

Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.

One-third of lung malignancies demonstrate a proneural/neuroendocrine phenotype or type of differentiation. However, it has not been clearly elucidated how proneural/neuroendocrine differentiation is controlled in lung cancers. We recently demonstrated that the POU3F2 gene plays a significant role in proneural/neuroendocrine differentiation of lung cancers. Because class III POU genes (POU3F1, POU3F2, POU3F3, and POU3F4) and class IV POU genes (POU4F1, POU4F2, and POU4F3) share similar properties in neural development, we analyzed the association between class III/IV POU genes and a proneural/neuroendocrine phenotype in lung cancers using seven small cell lung cancer (SCLC) cell lines and twelve non-SCLC (NSCLC) cell lines. Class III/IV POU gene expression was generally restricted to SCLC cells. However, the forced expression of class III/IV POU genes in the NSCLC cell lines induced the expression of neuroendocrine-specific markers (neural call adhesion molecule 1, synaptophysin, and chromogranin A) and proneural transcription factors (achaete-scute homolog-like 1, NeuroD1, and thyroid transcription factor 1) in various degrees. Furthermore, each class III/IV POU gene induced other class III/IV POU genes, suggesting the mutual induction of class III/IV POU genes. These findings suggest that the expression of class III/IV POU genes is important for the proneural/neuroendocrine differentiation of lung cancer cells.

Expression of developing neural transcription factors in lung carcinoid tumors.

In lung tumors, the association between carcinoids and high-grade neuroendocrine tumors (HGNETs) is controversial. To understand the phenotypic similarities/differences between lung carcinoids and HGNETs, we comparatively investigated the expression of three kinds of developing neural transcription factors (DNTFs: BRN2, TTF1 and ASCL1) and multiple endocrine neoplasia type 1 (MEN1) as well as RB1 and P53 using 18 carcinoids and 16 HGNETs. The DNTFs were expressed in 10 of the 18 carcinoids and in all the HGNETs, while normal neuroendocrine cells, which are considered the major cell origin of lung carcinoids and small cell carcinomas, did not express DNTFs. Both the DNTF(-) and DNTF(+) carcinoids contained typical and atypical carcinoids. All the DNTF(-) carcinoids examined were formed in the bronchial wall. All the MEN1(-) carcinoids examined were classified into the DNTF(-) carcinoids, while all the HGNETs expressed MEN1. This finding suggests that DNTF(-) MEN1(-) carcinoids are unlikely to be precursors of HGNETs. Although the status of RB1 and P53 between carcinoids and HGNETs were apparently different, the DNTF(+) carcinoids of two male patients and one female patient revealed morphologies resembling HGNET cells and relatively high Ki67 indices. Further investigation of DNTF expression in carcinoids might provide important clues to understand the association between carcinoids and HGNETs.

Prenatal diagnosis of umbilical artery thrombosis.

Umbilical artery thrombosis (UAT) is rare and few prenatally diagnosed cases have been reported. We describe 2 cases of fetal growth restriction prenatally diagnosed as UAT by ultrasound examination. In each case the cross section of the umbilical cord showed one normal artery and a small echogenic area which was suspected as an occluded thrombotic artery and they were surrounded by a highly curving 'C-shaped' vein. UAT was confirmed by histological examinations after deliveries in both cases. The characteristic ultrasound finding of the umbilical vessel, which is the so-called 'orange grabbed sign', enables the prenatal diagnosis of UAT and it is valuable with respect to perinatal fetal management because UAT is associated with increased perinatal morbidity and mortality.

Development of the human medullary arcuate nucleus from mid-gestation to the perinatal period: A morphometric study.

INTRODUCTION: Development of the human medullary arcuate nucleus (AN) has not been sufficiently investigated. The present study provides morphometric data by examining the brains from preterm and perinatal infants. MATERIALS AND METHODS: Nine brains were obtained from infants aged 21-43 postmenstrual weeks (PW). Serial celloidin sections were cut and stained using the Klüver-Barrera method. After microscopic observations, morphometric parameters [AN volume, numerical density (Nv) and total number (Nt) of neurons, and neuronal profile area (PA)] were analyzed. RESULTS: The AN was found as a pair of neuronal masses on the ventral medullary surface at 21 PW. Caudally, it was ventrolateral to the pyramidal tract (PT), and rostrally, medial to the PT. In the middle, it was diminished in size or interrupted. The AN neurons were gradually enlarged with age, showing multiplicity in size and shape. The following findings had a marked asymmetry and individual variability: (1) complete or partial inclusion of the AN in the PT; (2) connection between the rostral AN and the pontine nuclei; (3) coexistence of pyknotic neurons. The AN volume increased exponentially with age, while the Nv decreased exponentially. The Nt changed along two phases (decrease-increase) after mid-gestation. The mean PA increased linearly with age. Asymmetry and/or individual variability were demonstrated in the AN volume, Nt, and mean PA. CONCLUSIONS: Asymmetry and individual variability in the AN morphology are present in fetal period. The AN may undergo neuron death and neuroblasts production in tandem after mid-gestation.

Neural lineage-specific homeoprotein BRN2 is directly involved in TTF1 expression in small-cell lung cancer.

Thyroid transcription factor 1 (TTF1) plays crucial roles in thyroid, lung, and developing brain morphogenesis. Because TTF1-expressing neoplasms are generated from organs and tissues that normally express TTF1, such as the thyroid follicular epithelium and peripheral lung airway epithelium, TTF1 is widely used as a cell lineage-specific and diagnostic marker for thyroid carcinomas and for lung adenocarcinomas with terminal respiratory unit (TRU) differentiation. However, among lung neuroendocrine tumors, small-cell carcinomas (small-cell lung cancers (SCLCs)), most of which are generated from the central airway, also frequently express TTF1 at high levels. To clarify how SCLCs express TTF1, we investigated the molecular mechanisms of its expression using cultivated lung cancer cells and focusing upon neural cell-specific transcription factors. Both SCLC cells and lung adenocarcinoma cells predominantly expressed isoform 2 of TTF1, and TTF1 promoter assays in SCLC cells revealed that the crucial region for activation of the promoter, which is adjacent to the transcription start site of TTF1 isoform 2, has potent FOX-, LHX-, and BRN2-binding sites. Transfection experiments using expression vectors for FOXA1, FOXA2, LHX2, LHX6, and BRN2 showed that BRN2 substantially upregulated TTF1 expression, whereas FOXA1/2 weakly upregulated TTF1 expression. BRN2 and FOXA1/2 binding to the TTF1 promoter was confirmed through chromatin immunoprecipitation experiments, and TTF1 expression in SCLC cells was considerably downregulated after BRN2 knockdown. Furthermore, the TTF1 promoter in SCLC cells was scarcely methylated, and immunohistochemical examinations using a series of primary lung tumors indicated that TTF1 and BRN2 were coexpressed only in SCLC cells. These findings suggest that TTF1 expression in SCLC is a cell lineage-specific phenomenon that involves the developing neural cell-specific homeoprotein BRN2.

POU domain transcription factor BRN2 is crucial for expression of ASCL1, ND1 and neuroendocrine marker molecules and cell growth in small cell lung cancer.

BRN2 is a developmental neural cell-specific POU domain transcription factor and is crucial for cell lineage determination. We investigated the importance of BRN2 in the expression of the lineage-specific transcription factors (achaete-scute homolog-like 1 (ASCL1) and NeuroD1 (ND1)) and neural/neuroendocrine marker molecules (neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYP) and chromogranin A (CHGA)) in small cell lung cancer (SCLC) using cultured lung cancer cells. All examined SCLC cell lines expressed BRN2, as well as ASCL1, ND1, NCAM1, SYP and CHGA. The expression levels of ASCL1, ND1, NCAM1, SYP and CHGA considerably decreased when BRN2 was knocked down in SCLC cells, and the addition of a BRN2 transgene into non-SCLC (NSCLC) cells induced the expression of ASCL1, ND1, NCAM1, SYP and CHGA. However, the BRN2 gene was not activated by the forced expression of ASCL1 or ND1 in NSCLC cells. The knockdown of BRN2 caused significant growth retardation with decrease of S to G2 phase population and mitotic cell rates and unaltered Ki-67-labeled or apoptotic cell rates in SCLC cells, indicating increase of G1 phase population. These findings suggest that BRN2 is a higher level regulator than ASCL1 and ND1 and BRN2 might be involved in aggressiveness of SCLC.

Unencapsulated thymoma: a case report.

Thymomas, the most common mediastinal tumors, form capsules. Only a few reports have presented small thymomas without capsule formation, so-called microthymomas. Here, we report a case of an unencapsulated thymoma measuring 18 mm. A 42-year-old female presented with an anterior mediastinal tumor. Computed tomography revealed an 18-mm nodule in the anterior mediastinum. Magnetic resonance imaging revealed a solid tumor that was iso-intense on T1-weighted images and hypo-intense on T2-weighted images. Thoracoscopic partial thymectomy was performed. The histopathological diagnosis was a type B1 thymoma. The tumor was localized within the thymic tissue lacked a fibrous capsule and partially invaded the surrounding fat tissue. To our knowledge, this is the first report of an unencapsulated thymoma, except for microthymomas.

Multilayered Gel-Spotting Device for In Vitro Reconstruction of Hair Follicle-like Microstructure.

Hair follicles play an important role in hair development. This study aimed to develop a microgel-spotting device to fabricate a multilayered gel bead culture model and to mimic the early development of skin appendages to regenerate hair follicles in vitro. The model consists of an alginate gel layer containing cytokines as the core layer, a collagen gel layer containing mouse embryonic stem cells as the middle layer, and a collagen gel layer containing fetus-derived epidermal cells as the outer layer. A concentration gradient of cytokines is formed, which promotes interactions between epidermal and stem cells. Histological and immunnohistological analyses confirmed the reconstruction of hair follicle structures. As a result, the cell number and gel bead size could be precisely controlled by the developed microgel-spotting device. In the multilayered gel bead, the embryonic and epidermal cells cultured with the cytokine gradient formed cell aggregates with keratinized tissue in the center similar to "native" hair follicle structure. Sweat gland-like luminal tissue and erector pilorum-like structures were also observed around aggregates with concentric structures. In conclusion, the multilayered gel bead culture model demonstrated potential for in vitro hair follicle regeneration. The findings of this study provide insight into the early development of skin appendages.

Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin, and chromogranin A in lung cancer cells.

Neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYPT), and chromogranin A (CGA) are immunohistochemical markers for diagnosing lung neuroendocrine tumors (LNETs). However, the precise expression mechanisms have not been studied in enough detail. The purpose of the present study is to define the molecular mechanisms of NCAM1, SYPT, and CGA gene expressions, using cultivated lung cancer cells and focusing upon NeuroD1 (ND1), achaete-scute homolog-like 1 (ASCL1), and known transcription factors, repressor element 1 (RE1)-silencing transcription factor (REST) and c-AMP responsive element-binding protein (CREB). Promoter assays, chromatin immunoprecipitation, and transfection experiments revealed that ND1 activated NCAM1, that ASCL1 weakly upregulated SYPT expression, and that CGA expression was not regulated by ND1 or ASCL1. REST expression was restricted in non-small cell lung cancer (NSCLC) cells, and knockdown of REST could cause as much SYPT expression as in SCLC cells and weak CGA expression in NSCLC cells. However, CGA gene upregulation via CREB activation was not found in REST-lacking NSCLC cells, indicating the requirement of some additional mechanism for sufficient expression. These results suggest that NCAM1, SYPT and CGA expressions are differently regulated by neuroendocrine phenotype-specific transcription factors and provide a reason why NCAM1 and SYPT are frequently expressed in LNETs, irrespective of malignancy grade.