Stage III Chorioamnionitis is Associated with Reduced Risk of Severe Retinopathy of Prematurity.

OBJECTIVE: To identify whether histologically confirmed chorioamnionitis (hCAM) is associated with development of retinopathy of prematurity (ROP). STUDY DESIGN: We retrospectively analyzed 2 different cohorts. Cohort 1 was the national database of newborns in Japan born at ≤1500g or <32 weeks' gestation (January 2003 through April 2021, n = 38 013). Cohort 2 was babies born at <1500g from a single institution in Tsuchiura, Japan, (April 2015 through March 2018, n = 118). RESULTS: For Cohort1, after adjusting for potential confounders, stage III CAM (n = 5554) was associated with lower odds of severe ROP (stage ≥3 or required peripheral retinal ablation) by 14% (OR: 0.86; 95% CI: 0.78-0.94]. CAM of stage I (n = 3277) and II (n = 4319) was not associated with the risk of ROP. For Cohort 2, the odds of severe ROP were significantly reduced in moderate to severe hCAM groups (stage II, OR: 0.06, 95% CI: 0.05-0.82; stage III, OR: 0.10, 95% CI: 0.01-0.84). Neonates with funisitis, comorbidity of hCAM, and a finding of fetal inflammatory response had lower odds of severe ROP (OR: 0.11; 95% CI: 0.01-0.93). CONCLUSIONS: After adjusting for confounders, severe hCAM with fetal inflammatory response was associated with reduced risk of ROP.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Transcriptome analysis of umbilical cord mesenchymal stem cells revealed fetal programming due to chorioamnionitis.

Although chorioamnionitis (CAM) has been demonstrated to be associated with numerous short- and long-term morbidities, the precise mechanisms remain unclear. One of the reasons for this is the lack of appropriate models for analyzing the relationship between the fetal environment and chorioamnionitis and fetal programming in humans. In this study, we aimed to clarify the fetal programming caused by CAM using the gene expression profiles of UCMSCs. From nine preterm neonates with CAM (n = 4) or without CAM (n = 5), we established UCMSCs. The gene expression profiles obtained by RNA-seq analysis revealed distinctive changes in the CAM group USMSCs. The UCMSCs in the CAM group had a myofibroblast-like phenotype with significantly increased expression levels of myofibroblast-related genes, including α-smooth muscle actin (p < 0.05). In the pathway analysis, the genes involved in DNA replication and G1 to S cell cycle control were remarkably decreased, suggesting that cellular proliferation was impaired, as confirmed by the cellular proliferation assay (p < 0.01-0.05). Pathway analysis revealed that genes related to white fat cell differentiation were significantly increased. Our results could explain the long-term outcomes of patients who were exposed to CAM and revealed that UCMSCs could be an in vitro model of fetal programming affected by CAM.

Identification and validation of DNA methylation changes in pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) is a dangerous placental condition that can lead to premature labour, seizures and death of mother and infant. Several studies have identified altered placental DNA methylation in PE; however, there is widespread inconsistency between studies and most findings have not been replicated. This study aimed to identify and validate consistent differences in methylation across multiple PE cohorts. METHODS: Seven publicly available 450K methylation array datasets were analysed to identify consistent differentially methylated positions (DMPs) in PE. DMPs were identified based on methylation difference (≥10%) and significance (p-value ≤ 1 × 10-7). Targeted deep bisulfite sequencing was then performed to validate a subset of DMPs in an additional independent PE cohort. RESULTS: Stringent analysis of the seven 450K datasets identified 25 DMPs (associated with 11 genes) in only one dataset. Using more relaxed criteria confirmed 19 of the stringent 25 DMPs in at least four of the remaining six datasets. Targeted deep bisulfite sequencing of eight DMPs (associated with three genes; CMIP, ST3GAL1 and DAPK3) in an independent PE cohort validated two DMPs in the CMIP gene. Seven additional CpG sites in CMIP were found to be significantly differentially methylated in PE. DISCUSSION: The identification and validation of significant differential methylation in CMIP suggests that the altered DNA methylation of this gene may be associated with the pathogenesis of PE, and may have the potential to serve as diagnostic biomarkers for this dangerous condition of pregnancy.

The Fate of Transplanted Periodontal Ligament Stem Cells in Surgically Created Periodontal Defects in Rats.

Periodontal disease is chronic inflammation that leads to the destruction of tooth-supporting periodontal tissues. We devised a novel method ("cell transfer technology") to transfer cells onto a scaffold surface and reported the potential of the technique for regenerative medicine. The aim of this study is to examine the efficacy of this technique in periodontal regeneration and the fate of transplanted cells. Human periodontal ligament stem cells (PDLSCs) were transferred to decellularized amniotic membrane and transplanted into periodontal defects in rats. Regeneration of tissues was examined by microcomputed tomography and histological observation. The fate of transplanted PDLSCs was traced using PKH26 and human Alu sequence detection by PCR. Imaging showed more bone in PDLSC-transplanted defects than those in control (amnion only). Histological examination confirmed the enhanced periodontal tissue formation in PDLSC defects. New formation of cementum, periodontal ligament, and bone were prominently observed in PDLSC defects. PKH26-labeled PDLSCs were found at limited areas in regenerated periodontal tissues. Human Alu sequence detection revealed that the level of Alu sequence was not increased, but rather decreased. This study describes a novel stem cell transplantation strategy for periodontal disease using the cell transfer technology and offers new insight for cell-based periodontal regeneration.

Neuroprotective effects of human umbilical cord-derived mesenchymal stem cells on periventricular leukomalacia-like brain injury in neonatal rats.

BACKGROUND: Periventricular leukomalacia (PVL) is a type of multifactorial brain injury that causes cerebral palsy in premature infants. To date, effective therapies for PVL have not been available. In this study, we examined whether mesenchymal stem cells (MSCs) possess neuroprotective property in a lipopolysaccharide (LPS)-induced neonatal rat PVL-like brain injury. METHODS: Human umbilical cord-derived MSCs (UCMSCs) were used in this study. Four-day-old rats were intraperitoneally injected with LPS (15 mg/kg) to cause the PVL-like brain injury and were treated immediately after the LPS-injection with UCMSCs, conditioned medium prepared from MSCs (UCMSC-CM) or interferon-gamma (IFN-γ)-pretreated MSC (IFN-γ-UCMSC-CM). To assess systemic reaction to LPS-infusion, IFN-γ in sera was measured by ELISA. The brain injury was evaluated by immunostaining of myelin basic protein (MBP) and caspase-3. RT-PCR was used to quantitate pro-inflammatory cytokine levels in the brain injury, and the expression of tumor necrosis factor-stimulated gene-6 (TSG-6) or indoleamine 2,3-dioxygenase (IDO) to evaluate anti-inflammatory or immunomodulatory molecules in UCMSCs, respectively. A cytokine and growth factor array was employed to investigate the cytokine secretion profiles of UCMSCs. RESULTS: Elevated serum IFN-γ was observed in LPS-infused rats. The expression of IL-6, tumor necrosis factor-alpha (TNF-α), IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) were increased in the brain by LPS-infusion in comparison to saline-infused control. LPS-infusion increased caspase-3-positive cells and decreased MBP-positive area in neonatal rat brains. A cytokine and growth factor array demonstrated that UCMSCs secreted various cytokines and growth factors. UCMSCs significantly suppressed IL-1ß expression in the brains and reversed LPS-caused decrease in MBP-positive area. UCMSC-CM did not reverse MBP-positive area in the injured brain, while IFN-γ-UCMSC-CM significantly increased MBP-positive area compared to control (no treatment). IFN-γ-pretreatment increased TSG-6 and IDO expression in UCMSCs. CONCLUSION: We demonstrated that bolus intraperitoneal infusion of LPS caused PVL-like brain injury in neonatal rats and UCMSCs infusion ameliorated dysmyelination in LPS-induced neonatal rat brain injury. Conditioned medium prepared from IFN-γ-pretreated UCMSCs significantly reversed the brain damage in comparison with UCMSC-CM, suggesting that the preconditioning of UCMSCs would improve their neuroprotective effects. The mechanisms underline the therapeutic effects of MSCs on PVL need continued investigation to develop a more effective treatment.

Cell transfer technology for tissue engineering.

We recently developed novel cell transplantation method "cell transfer technology" utilizing photolithography. Using this method, we can transfer ex vivo expanded cells onto scaffold material in desired patterns, like printing of pictures and letters on a paper. We have investigated the possibility of this novel method for cell-based therapy using several disease models. We first transferred endothelial cells in capillary-like patterns on amnion. The transplantation of the endothelial cell-transferred amnion enhanced the reperfusion in mouse ischemic limb model. The fusion of transplanted capillary with host vessel networks was also observed. The osteoblast- and periodontal ligament stem cell-transferred amnion were next transplanted in bone and periodontal defects models. After healing period, both transplantations improved the regeneration of bone and periodontal tissues, respectively. This method was further applicable to transfer of multiple cell types and the transplantation of osteoblasts and periodontal ligament stem cell-transferred amnion resulted in the improved bone regeneration compared with single cell type transplantation. These data suggested the therapeutic potential of the technology in cell-based therapies for reperfusion of ischemic limb and regeneration of bone and periodontal tissues. Cell transfer technology is applicable to wide range of regenerative medicine in the future.

Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis.

BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo. METHODS: MSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model. RESULTS: PlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model. CONCLUSIONS: PlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic diseases.

Double-layered cell transfer technology for bone regeneration.

For cell-based medicine, to mimic in vivo cellular localization, various tissue engineering approaches have been studied to obtain a desirable arrangement of cells on scaffold materials. We have developed a novel method of cell manipulation called "cell transfer technology", enabling the transfer of cultured cells onto scaffold materials, and controlling cell topology. Here we show that using this technique, two different cell types can be transferred onto a scaffold surface as stable double layers or in patterned arrangements. Various combinations of adherent cells were transferred to a scaffold, amniotic membrane, in overlapping bilayers (double-layered cell transfer), and transferred cells showed stability upon deformations of the material including folding and trimming. Transplantation of mesenchymal stem cells from periodontal ligaments (PDLSC) and osteoblasts, using double-layered cell transfer significantly enhanced bone formation, when compared to single cell type transplantation. Our findings suggest that this double-layer cell transfer is useful to produce a cell transplantation material that can bear two cell layers. Moreover, the transplantation of an amniotic membrane with PDLSCs/osteoblasts by cell transfer technology has therapeutic potential for bone defects. We conclude that cell transfer technology provides a novel and unique cell transplantation method for bone regeneration.

Placenta Mesenchymal Stem Cell Derived Exosomes Confer Plasticity on Fibroblasts.

Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc.

Detection of ALK rearrangement in an octogenarian patient with pleomorphic carcinoma of the lung.

We herein present a rare case of ALK-positive pulmonary pleomorphic carcinoma in an octogenarian patient. A computed tomography scan of the thorax indicated a pulmonary nodule in the right upper lobe of an asymptomatic 87-year-old female. The surgical resection revealed that the disease was pleomorphic carcinoma with pathological T2aN0M0, stage IB. EML4-ALK was evaluated using immunohistochemistry and fluorescence in situ hybridization, and EGFR mutations were analyzed using the Cycleave method. While there were no EGFR mutations detected, she was positive for the ALK rearrangement. This is the first report of ALK rearrangement in an octogenarian patient with pleomorphic carcinoma of the lung.

Periodontal regeneration using periodontal ligament stem cell-transferred amnion.

Periodontal disease is characterized by the destruction of tooth supporting tissues. Regeneration of periodontal tissues using ex vivo expanded cells has been introduced and studied, although appropriate methodology has not yet been established. We developed a novel cell transplant method for periodontal regeneration using periodontal ligament stem cell (PDLSC)-transferred amniotic membrane (PDLSC-amnion). The aim of this study was to investigate the regenerative potential of PDLSC-amnion in a rat periodontal defect model. Cultured PDLSCs were transferred onto amniotic membranes using a glass substrate treated with polyethylene glycol and photolithography. The properties of PDLSCs were investigated by flow cytometry and in vitro differentiation. PDLSC-amnion was transplanted into surgically created periodontal defects in rat maxillary molars. Periodontal regeneration was evaluated by microcomputed tomography (micro-CT) and histological analysis. PDLSCs showed mesenchymal stem cell-like characteristics such as cell surface marker expression (CD90, CD44, CD73, CD105, CD146, and STRO-1) and trilineage differentiation ability (i.e., into osteoblasts, adipocytes, and chondrocytes). PDLSC-amnion exhibited a single layer of PDLSCs on the amniotic membrane and stability of the sheet even with movement and deformation caused by surgical instruments. We observed that the PDLSC-amnion enhanced periodontal tissue regeneration as determined by micro-CT and histology by 4 weeks after transplantation. These data suggest that PDLSC-amnion has therapeutic potential as a novel cell-based regenerative periodontal therapy.

Pneumocystis jiroveci pneumonia in a patient with rheumatoid arthritis as a complication of treatment with infliximab, anti-tumor necrosis factor alpha neutralizing antibody.

We report that a-63-year-old woman developed Pneumocystis jiroveci pneumonia (PCP) as a complication from treatment with infliximab for rheumatoid arthritis. Although there was neither symptoms of dyspnea nor typical observations on a chest X-ray examination, low levels of oxygen saturation and findings of high-resolution chest computed tomographic scanning suggested a possibility of interstitial pneumonia. A polymerase chain reaction-based detection of Pneumocystis jiroveci in induced sputum allowed an early diagnosis of PCP and subsequent effective treatment.

Usefulness of endobronchial ultrasonography for transbronchial lung biopsies of peripheral lung lesions.

BACKGROUND: Peripheral lung lesions are increasing in numbers. Endoscopic diagnosis is essential for the prevention of unnecessary operations. Conventional diagnostic procedures have limitations in availability and results. OBJECTIVES: Endobronchial ultrasonography (EBUS) was investigated as a means to guide transbronchial lung biopsy, to reduce the discomfort during the procedure and to improve diagnostic accuracy. METHODS: In 50 cases, we performed transbronchial lung biopsy combined with EBUS and fluoroscopic guidance. The results were compared to 42 controls assessed by fluoroscopy only. RESULTS: In 38 cases (76%), EBUS could describe the peripheral lesion (33 from inside, including 9 cases with difficulties in fluoroscopic observation, and 5 from an adjacent bronchus, indicating the correct location of the lesion). If successfully placed inside, a change in the patient's position was not required, which helped to reduce patient discomfort. Lung cancer was diagnosed in 24 patients and benign disease in 25 patients; in 1 case diagnosis remained unknown. When the EBUS probe could be introduced inside the lesion, the sensitivity for cancer diagnosis and specificity for cancer exclusion were 100%, respectively (15/15, 18/18). Compared to the controls in whom the biopsy site was determined by fluoroscopy only, the sensitivity tended to be superior by EBUS, although it did not reach statistical significance (p = 0.06). However, specificity and accuracy were statistically significant (both p = 0.02). CONCLUSIONS: When the lesion can be correctly described by EBUS from inside the lesion, EBUS is useful to guide transbronchial lung biopsy, can contribute to a reduction in patient discomfort and improves the accuracy of diagnosis. Additional navigation tools to increase correct positioning of the EBUS probe are desirable.