Cyclophosphamide exposure factors in family caregivers for pediatric cancer patients.

The exposure of family caregivers to anticancer drugs for pediatric patients with malignancy is a potential health risk that needs to be minimized. We monitored the amount of cyclophosphamide (CPM) that had adhered to the undershirts of patients and the personal protective equipment (PPE) of family caregivers as well as the caregivers' urine levels of CPM within the first three days after the first and second courses of high-dose CPM therapy. Liquid chromatography/mass spectrometry (LC/MS/MS) detected >0.03 ng/ml of CPM in 26% (23/88) of urine samples from 8 of 11 (72.7%) patients' family caregivers, with a peak of 0.7 ng/ml from 24 to 48 h after administration. Since urine CPM concentrations in family caregivers varied after the first and second courses, the exposure risk factors were analyzed by scoring the PPE-wearing time index (caring minutes × PPE points from wearing masks, gloves, and/or gowns) and CPM adhesion of PPE items with the caring patterns of diaper change, washing body care, oral care, eating assistance, emotional support, and co-sleeping. The closest association was observed for CPM adhesion between oral care gloves and undershirts (correlation coefficient 0.67, p = 0.001). The mixed-effect model analysis indicated only a significant correlation between the PPE-wearing time index and emotional care (playing, cuddling, and physical contact) (p = 0.016). These results suggest that prolonged emotional support results in poor PPE protection, which increases the risk of exposure in family caregivers. Strict PPE care within 48 h after high-dose CPM controls the exposure to high-risk anticancer drugs in caregivers of pediatric patients.

Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice.

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-ß1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-ß1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-ß1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease.

Sarcoidosis and sarcoidal foreign body reaction after permanent eye makeup application: Analysis by immunohistochemistry with commercially available antibodies specific to Cutibacterium acnes and Mycobacteria.

We report two cases of eyebrow granulomas in patients who underwent a permanent eye makeup procedure. A rash was observed 16 months after the procedure in Case 1, and 10 years after the procedure in Case 2. Histopathologically, both patients exhibited noncaseating epithelioid cell granulomas. In Case 1, most of the black-brown granules of the permanent makeup were not present in the granulomas but were localized in the upper dermis. In contrast, in Case 2, some of the black-brown granules were phagocytized in the granulomas, preferentially within the giant cells. Based on systemic examinations, the patients from Cases 1 and 2 were diagnosed with sarcoidosis and sarcoidal foreign body reaction, respectively. To clarify the pathogenesis of our cases, we performed immunohistochemistry using commercially available monoclonal antibodies specific to Cutibacterium acnes, previously Propionibacterium acnes (PAB), and Mycobacteria (LAM antibody). PAB antibody results were positive in granulomas only in Case 1, and the LAM antibody results were negative in both cases. Immunohistochemical detection of C. acnes in granulomas could provide useful information for differentiating between cutaneous sarcoidosis and sarcoidal foreign body reactions.

Chronic spinal cord injury functionally repaired by direct implantation of encapsulated hair-follicle-associated pluripotent (HAP) stem cells in a mouse model: Potential for clinical regenerative medicine.

Chronic spinal cord injury (SCI) is a highly debilitating and recalcitrant disease with limited treatment options. Although various stem cell types have shown some clinical efficacy for injury repair they have not for SCI. Hair-follicle-associated pluripotent (HAP) stem cells have been shown to differentiate into neurons, Schwan cells, beating cardiomyocytes and many other type of cells, and have effectively regenerated acute spinal cord injury in mouse models. In the present report, HAP stem cells from C57BL/6J mice, encapsulated in polyvinylidene fluoride membranes (PFM), were implanted into the severed thoracic spinal cord of C57BL/6J or athymic nude mice in the early chronic phase. After implantation, HAP stem cells differentiated to neurons, astrocytes and oligodendrocytes in the regenerated thoracic spinal cord of C57BL/6J and nude mice. Quantitative motor function analysis, with the Basso Mouse Scale for Locomotion (BMS) score, demonstrated a significant functional improvement in the HAP-stem-cell-implanted mice, compared to non-implanted mice. HAP stem cells have critical advantages over other stem cells: they do not develop teratomas; do not loose differentiation ability when cryopreserved and thus are bankable; are autologous, readily obtained from anyone; and do not require genetic manipulation. HAP stem cells therefore have greater clinical potential for SCI repair than induced pluripotent stem cells (iPSCs), neuronal stem cells (NSCs)/neural progenitor cells (NPCs) or embryonic stem cells (ESCs). The present report demonstrates future clinical potential of HAP-stem-cell repair of chronic spinal cord injury, currently a recalcitrant disease.

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Can Extensively Differentiate to Tyrosine-Hydroxylase-Expressing Dopamine-Secreting Neurons.

Hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of hair follicles from mice and humans and have been shown to differentiate to neurons, glia, keratinocytes, smooth muscle cells, melanocytes and beating cardiac muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal-cord regeneration in mouse models, differentiating to Schwann cells and neurons in this process. HAP stem cells can be banked by cryopreservation and preserve their ability to differentiate. In the present study, we demonstrated that mouse HAP stem cells cultured in neural-induction medium can extensively differentiate to dopaminergic neurons, which express tyrosine hydroxylase and secrete dopamine. These results indicate that the dopaminergic neurons differentiated from HAP stem cells may be useful in the future to improve the symptoms of Parkinson's disease in the clinic.

Mothers' Experiences With Pregnancy and Childbirth Following Pediatric Living Liver Transplant Donation: A Qualitative Descriptive Study.

BACKGROUND: Half of pediatric living liver transplantation donors are mothers, including women of reproductive age. Reports on pregnancy and childbirth after living donor liver transplantation are limited to medical aspects, and mothers' experiences remain unclear. We describe the experiences of women who became pregnant and gave birth after living donor liver transplantation. METHODS: We used a qualitative descriptive approach. Eleven women who became pregnant and delivered following pediatric living liver transplant donation participated in face-to-face, in-depth interviews. Data collected via semi-structured interviews were assessed using an inductive qualitative analysis. The study was conducted in accordance with the Declaration of Helsinki. RESULTS: Women's experiences with pregnancy and childbirth following pediatric living liver transplant donation were categorized as follows: explanation and consultation on pregnancy and childbirth after liver donation; physical and mental burden after liver donation; concern about the effects of donor surgery on pregnancy and childbirth; consideration for own body; concern about the physical condition of my child, who is the recipient; and the presence of health professionals with which to easily consult. CONCLUSION: After donation, mothers are physically burdened and experiences anxiety about the physical condition of the recipient as well as about pregnancy and childbirth. Therefore, continuous psychosocial support is necessary.

Expression of anti-aging type-XVII collagen (COL17A1/BP180) in hair follicle-associated pluripotent (HAP) stem cells during differentiation.

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.

Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Encapsulated on Polyvinylidene Fluoride Membranes (PFM) Promote Functional Recovery from Spinal Cord Injury.

Our previous studies showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells, which reside in the bulge area of the hair follicle, could restore injured nerve and spinal cord and differentiate into cardiac muscle cells. Here we transplanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell colonies enclosed on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord of nude mice. After seven weeks of implantation, we found the differentiation of HAP stem cells into neurons and glial cells. Our results also showed that PFM-captured GFP-expressing HAP stem-cell colonies assisted complete reattachment of the thoracic spinal cord. Furthermore, our quantitative motor function analysis with the Basso Mouse Scale for Locomotion (BMS) score demonstrated a significant improvement in the implanted mice compared to non-implanted mice with a severed spinal cord. Our study also showed that it is easy to obtain HAP stem cells, they do not develop teratomas, and do not loose differentiation ability when cryopreserved. Collectively our results suggest that HAP stem cells could be a better source compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells for regenerative medicine, specifically for spinal cord repair.

Implanted hair-follicle-associated pluripotent (HAP) stem cells encapsulated in polyvinylidene fluoride membrane cylinders promote effective recovery of peripheral nerve injury.

Hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area of the hair follicle, express the stem-cell marker, nestin, and have been shown to differentiate to nerve cells, glial cells, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes. Transplanted HAP stem cells promote the recovery of peripheral nerve and spinal cord injuries and have the potential for heart regeneration as well. In the present study, we implanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell spheres encapsulated in polyvinylidene fluoride (PVDF)-membrane cylinders into the severed sciatic nerve of immunocompetent and immunocompromised (nude) mice. Eight weeks after implantation, immunofluorescence staining showed that the HAP stem cells differentiated into neurons and glial cells. Fluorescence microscopy showed that the HAP stem cell hair spheres promoted rejoining of the sciatic nerve of both immunocompetent and immunodeficient mice. Hematoxylin and eosin (H&E) staining showed that the severed scatic nerves had regenerated. Quantitative walking analysis showed that the transplanted mice recovered the ability to walk normally. HAP stem cells are readily accessible from everyone, do not form tumors, and can be cryopreserved without loss of differentiation potential. These results suggest that HAP stem cells may have greater potential than iPS or ES cells for regenerative medicine.

Cryopreservation of the Hair Follicle Maintains Pluripotency of Nestin-Expressing Hair Follicle-Associated Pluripotent Stem Cells.

Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. In the present study, we established efficient cryopreservation methods of the hair follicle that maintained the pluripotency of HAP stem cells. We cryopreserved the whole hair follicle from green fluorescent protein transgenic mice by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum (FBS). After 4 weeks of culture, cells from the upper part of the hair follicle grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week of culture, the growing cells formed hair spheres, each containing ∼1×10(2) HAP stem cells. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. In contrast, rapid-cooling (vitrification) cryopreservation poorly preserved the pluripotency of the hair follicle stem cells. Stem cell marker genes (nestin, Sox2, and SSEA-1) were as highly expressed in slow-rate cooled cryopreserved follicles, after thawing, as in fresh follicles. However, in the vitrification cryopreserved follicles, the expression of the stem cell marker genes was greatly reduced. Direct cryopreservation of hair spheres by either the rapid-cooling, or slow-cooling method, resulted in loss of pluripotency. These results suggest that the slow-rate cooling cryopreservation of the whole hair follicle is effective to store HAP stem cells. Stored HAP stem cells would be very useful in personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.

Cystatin SN upregulation in patients with seasonal allergic rhinitis.

Seasonal allergic rhinitis (SAR) to the Japanese cedar, Cryptomeria japonica (JC) pollen is an IgE-mediated type I allergy affecting nasal mucosa. However, the molecular events underlying its development remain unclear. We sought to identify SAR-associated altered gene expression in nasal epithelial cells during natural exposure to JC pollen. We recruited study participants in 2009 and 2010 and collected nasal epithelial cells between February and April, which is the period of natural pollen dispersion. Fifteen patients with SAR-JC and 13 control subjects were enrolled in 2009, and 17 SAR-JC patients, 13 sensitized asymptomatic subjects (Sensitized), and 15 control subjects were enrolled in 2010. Total RNA was extracted from nasal epithelial cells and 8 SAR-JC patients and 6 control subjects in 2009 were subjected to microarray analysis with the Illumina HumanRef-8 Expression BeadChip platform. Allergen-stimulated histamine release was examined in the peripheral blood basophils isolated from patients with SAR. We identified 32 genes with significantly altered expression during allergen exposure. One of these, CST1 encodes the cysteine protease inhibitor, cystatin SN. CST1 expression in nasal epithelial cells was significantly upregulated in both the 2009 and 2010 SAR-JC groups compared with the control groups. Immunohistochemical staining confirmed the increased expression of CST1 in the nasal epithelial cells of SAR patients. Addition of exogenous CST1 to basophils inhibited JC allergen-stimulated histamine release in vitro. We propose that CST1 may contribute to inactivation of protease allergens and help re-establish homeostasis of the nasal membranes.

Generation of induced pluripotent stem cells from human nasal epithelial cells using a Sendai virus vector.

The generation of induced pluripotent stem cells (iPSCs) by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs)-derived iPSCs by gene transduction with Sendai virus (SeV) vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08-0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes.

In vivo imaging of nuclear-cytoplasmic deformation and partition during cancer cell death due to immune rejection.

In this report, we investigated the in vivo cell biology of cancer cells during immune rejection. The use of nestin-driven green fluorescent protein (ND-GFP) transgenic mice as hosts, in which nascent blood vessels express GFP, and implanted dual-color mouse mammary tumor 060562 (MMT) cells, in which the cytoplasm expresses red fluorescent protein (RFP) and the nuclei express GFP, allowed very important novel observations of angiogenesis and subcellular death pathways during immune rejection of a tumor. Nascent blood vessels did not form in the initially-growing mouse mammary tumor in ND-GFP immunocompetent mice. In contrast, in ND-GFP immunodeficient nude mice, numerous GFP-expressing nascent blood vessels grew into the tumor. The results suggest that insufficient nascent tumor angiogenesis was important in tumor rejection. During immune rejection, the cancer cells deformed their cytoplasm and nuclei, which were readily imaged by RFP and GFP, respectively. The nuclear membrane of the cancer cells ruptured, and chromatin extruded during partition of cytoplasm and nuclei. T lymphocytes infiltrated into the initially-growing tumor in the nestin-GFP transgenic immunocompetent mice. The cytotoxic role of the sensitized T lymphocytes was confirmed in vitro when they were co-cultured with MMT cells. The CD8a-positive lymphocytes attached to the cancer cells and caused nuclear condensation, deformation, and partition from their cytoplasm, similar to what occurred in vivo. The color-coded subcellular fluorescence-imaging model of immune rejection of cancer cells can provide a comprehensive system for further testing of immune-based treatment for cancer.

Nestin-positive hair follicle pluripotent stem cells can promote regeneration of impinged peripheral nerve injury.

Nestin-positive, keratin 15 (K15)-negative multipotent hair follicle stem cells are located above the hair follicle bulge. We have termed this location the hair follicle pluripotent stem cell area. We have previously shown that transplantation of nestin-expressing hair follicle stem cells can regenerate peripheral nerve and spinal cord injuries. In the present study, we regenerated the impinged sciatic nerve by transplanting hair follicle pluripotent stem cells. Human hair follicle stem cells were transplanted around the impinged sciatic nerve of ICR nude (nu/nu) mice. The hair follicle stem cells were transplanted between impinged sciatic nerve fragments of the mouse where they differentiated into glial fibrillary acidic protein-positive Schwann cells and promoted the recovery of pre-existing axons. The regenerated sciatic nerve functionally recovered. These multipotent hair follicle stem cells thereby provide a potential accessible, autologous source of stem cells for regeneration therapy of nerves degenerated by compression between bony or other hard surfaces.

[A man with severe dilated cardiomyopathy with implantable cardioverter-defibrillator who underwent sigmoidectomy].

We report a man with severe dilated cardiomyopathy with an implantable cardioverter-defibrillator (ICD) who underwent sigmoidectomy. During the operation, the defibrillation function of the ICD has been stopped to prevent malfunction caused by electrocautery artifacts, and the electrodes of the external defibrillator were placed on the chest wall. Pulmonary artery catheter was inserted under X-ray imaging to prevent the interference between ICD leads and the catheter. Anesthesia was maintained with combined general and thoracic epidural anesthesia. In order to prevent the afterload increase, both milrinone and carperitide were administered. Fluid resuscitation was also performed to maintain circulating blood volume. As a result of the management, patient has not exhibited any heart failure.