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In Japan, 156 cases of dura mater-transplanted Creutzfeldt-Jakob disease(dCJD)with a history of Lyodura transplantation have been confirmed until February 2022, with only a few new cases still being identified. The history of Lyodura transplantation is one involving a neurosurgical procedure. The cumulative global number of cases of bovine spongiform encephalopathy-related variant CJD(BSE-related vCJD), which has shaken societies around the world, is 232 as of 2019. Thus, the impact of dCJD on the society in Japan needs no explanation. Thanks to the world's concerted efforts in research and countermeasures, medically induced prion diseases are finally becoming a thing of the past. However, due to the extremely long incubation period of CJD and the difficulty of tracing the source of infection, immediate action in the event of an outbreak is not possible, and efforts must focus on preventing disease outbreaks. Independent of this, approximately 200 cases of solitary and hereditary prion diseases occur annually in Japan. If neurosurgery must be performed on such patients, secondary transmission of prion disease by neurosurgical instruments must be prevented. Therefore, sterilization methods for neurosurgical instruments are critical, and various measures including sterilization methods have been determined and published by a research group designated by the Japanese Ministry of Health, Labour and Welfare. The sterilization of neurosurgical instruments should comply with the latest guidelines that are published by this study group.

BACKGROUND/AIM: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been affecting Hokkaido, Japan since late February 2020 until present. The aim of this study was to report the relationship between anti-SARS-CoV-2 antibody-positive and SARS-CoV-2 PCR-positive cases by analyzing anti-SARS-CoV-2 antibodies (IgG and total-Ig). PATIENTS AND METHODS: Serum samples were collected from care workers and nurses in two nursing homes and two hospitals which underwent virus outbreak. All people were confirmed to be SARS-CoV-2-positive by RT-qPCR and their sera was analyzed for anti-SARS-CoV-2 antibodies (IgG and total-Ig). RESULTS: Although 34 out of 43 samples (79.1%) showed enough amount of anti-SARS-CoV-2 antibodies, 9 RT-qPCR -positive samples (20.9%) showed absence of anti-SARS-CoV-2 antibodies in their sera. CONCLUSION: The results that 20.9% of RT-qPCR-positive samples with SARS-CoV-2 showed absence of anti-SARS-CoV-2 antibodies provides a possibility that the innate immune reaction could eliminate the virus without activating adaptive immune reaction.

Although epidemiological studies have shown a relationship between periodontal disease and pancreatic cancer, the molecular mechanisms involved remain unclear. In this study, the effects of systemic administration of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on gene expression were comprehensively explored in mouse pancreas that did not demonstrate any signs of inflammation. PG-LPS was prepared in physiological saline and intraperitoneally administered to male mice at a concentration of 5 mg/kg every 3 days for 1 month. After extracting total RNA from the excised mice pancreas, a comprehensive DNA microarray analysis of gene expression was performed. Tissue specimens were also subjected to hematoxylin&ndash, eosin staining and immunohistochemistry using anti-regenerating islet-derived 3A and G (Reg3A/G) antibody. ImageJ software was used to quantify the area of Reg3A/G positive cells in pancreatic islets by binarizing image date followed by area extraction. The results were compared using Mann&ndash, Whitney U test. Data are presented as mean &plusmn, standard deviation (SD) with p &lt, 0.05 considered as significant. Reg3G, a gene related to pancreatic cancer, was one of the 10 genes with the highest levels of expression in the pancreas stimulated with PG-LPS. The comprehensive analysis revealed a 73-fold increase in Reg3G expression level in the PG-LPS group when compared with the control group, in addition, the expression level of Reg3A was increased by 11-fold in the PG-LPS group. Image analysis showed that the ratio of Reg3A/G positive cells was higher in the PG-LPS group than the control. Immunostaining showed the presence of Reg3A/G-positive cells in the alpha-cell equivalent areas around the islets of Langerhans in the PG-LPS group. These results support the notion that periodontal disease may be a risk factor for pancreatic cancer.

Background In Japan, there have been approximately 50 000 deaths from gastric cancer annually for over 40 years with little variation. It has been reported that most gastric cancers in Japan are caused by Helicobacter pylori infection. H. pylori eradication therapy was approved for patients with chronic gastritis by the Japanese national health insurance scheme in February 2013 for patients with an endoscopic diagnosis of chronic gastritis is positive for H. pylori. We examined the effect on gastric cancer death rate 4 years after expansion of health insurance coverage. Aim We conducted an epidemiological study and analyzed trends in prescription for H. pylori eradication therapy. We used the electronic medical claims database from Hokkaido, Japan to evaluate the impact of expansion of national health insurance coverage for H. pylori eradication therapy on deaths from gastric cancer. Methods Data on deaths from gastric cancer were obtained from the Japanese Ministry of Health, Labour and Welfare and the Cancer Statistics in Japan (2015). Analysis of electronic claims records was performed using the National Database, mainly focusing on Hokkaido. Prescriptions for H. pylori eradication therapy and the number of patients treated for gastric cancer were also extracted from the Hokkaido database. Results Approximately 1.5 million prescriptions for H. pylori eradication therapy were written annually. Gastric cancer deaths fell each year: 48 427 in 2013, 47 903 in 2014, 46 659 in 2015, and 45 509 in 2016, showing a significant decrease after expansion of insurance coverage for H. pylori eradication therapy (P

This review provides a thorough and clear discussion on the outcomes of stem cells in treating chronic wounds. With recent technological developments that now allow isolation and culture of stem cells, researchers are able to perform vigorous studies on somatic or adult stem cells. Human and animal stem cell studies are discussed with a focus on the basic process of stem cells in wound healing and the authors' first-hand clinical experience with stem cells used for chronic wound healing., Clinics in Plastic Surgery, 39(3), pp.281-292; 2012

Color changes of visible and exposed body surfaces, such as the face and extremities, after burn injury or surgery, such as skin grafting, flap, or sclerotherapy for vascular malformations, are sometimes a concern. The consequences reduce the satisfaction of both patients and physicians. An easy and reproducible method has not yet been established for an objective analysis of color changes; therefore, we tested a hand-held color analyzer (NF-333; Nippon Denshoku Co. Ltd) with data transport to a computer database and analysis software for posttreatment skin color change. The parameters included L, a, and b, which measure clarity, red, and yellow, respectively. Two groups were prospectively divided with 20 (11 females and nine males) patients per group. One group received skin grafting plus basic fibroblast growth factor (bFGF) spray daily and the other group received only skin grafting. The patients were randomized by the date of their first visit to our hospital. Patients were treated with bFGF on odd days, while patients who came on even days were included in the non-bFGF-treated group. The donor site for skin grafting was the lateral thighs and the thickness was similar in both groups. The results were compared at 1-year posttreatment follow-up. Clinical and objective assessments of the scars were performed 1 to years after complete healing. Color change differentials in comparison with the surrounding skin were lower with bFGF treatment in all parameters (p

Systemic and local radiation injuries caused by nuclear power reactor accidents, therapeutic irradiation, or nuclear terrorism should be prevented or properly treated in order to improve wound management and save lives. Currently, regenerative surgical modalities should be attempted with temporal artificial dermis impregnated and sprayed with a local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Human mesenchymal stem cells and adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and were tested for differentiation and local stimulation effects in the radiation-exposed wounds. The perforator flap and artificial dermal template with growth factor were successful for reconstruction in patients who were suffering from complex underlying disease. Patients were uneventfully treated with minimal morbidities. In the experiments, the hMSCs are strongly proliferative even after 20 Gy irradiation in vitro. In vivo, 4 Gy rat whole body irradiation demonstrated that sustained marrow stromal (mesenchymal stem) cells survived in the bone marrow. Immediate artificial dermis application impregnated with cells and the cytokine over the 20 Gy irradiated skin and soft tissues demonstrated the significantly improved fat angiogenesis, architected dermal reconstitution, and less inflammatory epidermal recovery. Detailed understanding of underlying diseases and rational reconstructive procedures brings about good outcomes for difficult irradiated wound healing. Adipose-derived stem cells are also implicated in the limited local injuries for short cell harvesting and processing time in the same subject., Health Physics, 98(6), pp.858-862; 2010

BACKGROUND: The pathogenesis of keloid is poorly understood. Although vigorous investigations have attempted to elucidate the mechanisms or causative factors of keloid, there are little data on why keloids are very intractable and recur easily in each patient. METHODS: In an attempt to analyze the possible interaction between human mesenchymal stem cells and keloid-derived fibroblasts, the dual-chamber cell-migration assay, cell proliferation, ultrastructural morphology, and Western blot analysis were used to investigate the production of the extracellular matrices of the coculture. RESULTS: Cell proliferation was not significantly different between keloid-derived fibroblasts and normal dermal fibroblasts during a 4-day observation period. There was a significant cell migration of human mesenchymal stem cells when keloid-derived fibroblasts were placed in the bottom chamber, compared to when normal dermal fibroblasts were placed in the same way in 8-microm diameter pore membranes (190.6 +/- 51.45 and 32.0 +/- 6.20 cells/field, respectively, P < 0.01). With 3-microm diameter pores, the human mesenchymal stem cells migrated in the pores only when the keloid-derived fibroblasts were placed in the bottom chambers (6.4 +/- 3.84 cells/field). Monolayer coculture of human mesenchymal stem cells and keloid-derived fibroblasts demonstrated further functional differentiation, such as collagen secretion and abundant rough endoplasmic reticulum. Western blot analysis of the cells in the modified dual-chamber culture demonstrated most significantly abundant fibronectin expression when the human mesenchymal stem cells contained keloid fibroblasts. CONCLUSION: The results of this study may indicate that human mesenchymal stem cells participate and recruit in keloid pathogenesis by differentiating themselves toward keloid recalcitrant formation and progression., International Journal of Dermatology, 47(11), pp.1112-1117; 2008

Acute wounds caused by the severe and extensive infection such as alpha-hemolytic streptococci or MRSA may be life-threatening in immunocompromised hosts such as steroid users or those with venostasis in the lower legs. The principle of the treatment is the removal of the primary pathogens, however, difficulties of the lower leg reconstruction remain due to the tissue loss and the deficiency of the circulation system after extensive soft tissue debridement. For 10 immediately-developing extensive lower extremity cases, artificial skin substitutes composed of dried bilayer membranes of outer silicone membrane and inner porcine-tendon derived collagen layer (Pelnac®, Gunze Co., Ltd., Kyoto, Japan) were immediately after extensive and meticulous debridement. While the skin substitute integrated into the wound bed for maximally 3 weeks, total 20 micrograms of the basic fibroblast growth factors (bFGF)(Trafermin®, Kaken Pharmaceutical Co. Ltd, Tokyo, Japan) were applied by the 27-Gage syringe daily. The outer membranes were removed and thin partial thickness skin grafting (9–10/1,000 inches) was performed and wound healed uneventfully. The bFGF-treated skin texture was significantly softer compared to control by a durometer (Teclock®, GS-701N, Tokyo, Japan)(p

Various methods have been attempted for the treatment and management of keloids; however, there is little satisfactory clinical evidence in long-term follow ups. Also, there is a preference for occurrence and recurrence in anatomic location. Usually anatomic locations with higher regional tension and more sebaceous glands are inclined toward pathogenesis. Thirty-eight keloids treated with combined surgical excision and postoperative irradiation, using electron beams with only a 10-mm opening by lead shielding, were investigated at a mean follow up of 4.4 +/- 2.5 years (range, 1-9 years) at a single institute. Ten locations such as the ear (n = 6), neck (n = 3), and upper lip (n = 1) were among the craniofacial locations. The hardness of the keloids and posttreatment scars was clinically and objectively tested with the Vancouver scar scale and a durometer, which is often used for the industrial measurement of thread balls and rubber. At a mean of 4.4 +/- 2.5 years of follow up, the clinical characteristics of the scars were significantly better posttreatment as 2.6 +/- 0.5 versus 1.0 +/- 0.6, 3.7 +/- 0.7 versus 1.7 +/- 0.7, 2.9 +/- 0.4 versus 1.3 +/- 0.5, and 2.7 +/- 0.5 versus 1.3 +/- 0.5 (keloid scars versus posttreatment scars: pigmentation, pliability, height and vascularity, respectively, P < 0.01). The durometer readings were significantly lower posttreatment, 15.2 +/- 3.9 versus 7.7 +/- 2.9 (keloid scars versus posttreatment scars, P < 0.01). The recurrence rate was 21.2% overall with none in craniofacial locations. Therefore, the combined treatment of surgical excision and postoperative electron beam irradiation is effective for scar quality and reducing the recurrence rate in long-term follow up.

Skin and soft tissue radiation injuries are life-threatening as well as of esthetic concern in local tissues. As for local radiation injuries, clinically developed procedures such as local flaps and free vascularized flaps result in better and less-invasive reconstructive outcomes for victims of therapeutic radiation of malignant tumors, prolonged fluoroscopic procedures for cardiovascular diseases or possibly direct contact with radioactive waste, while systemic radiation injuries should find other solutions. In systemic radiation injuries, there is a good chance to save lives when the initial treatment is efficient, although donors of the skin grafting after debridement of the radiation-damaged tissue removal are unrealistically autogenous. Thus, exceeding certain extent and degree, systemic radiation injury-induced burns were not the medical objectives. Recent understanding and application of use in adult stem cells, however, create a new insight for severe systemic radiation injuries. Among the adult stem cells, the human mesenchymal stem cells are able to be freeze-dried, easily thawed and able to be cultured in vitro. A basic fibroblast growth factor is already of clinical use and efficient for the human mesenchymal stem cell growth both in vitro and in vivo. An artificial dermis is useful for temporal coverage of the raw surface of severe bedsores. The combined use of these successfully regenerates both dermis and epidermis after 20 Gy whole body irradiation in a nude rat model. Therefore, even severe radiation injuries, local tissue management and therapy are well-handled and systemic radiation injury may be hopeful with understanding and the specialized practice of regenerative medicine.

Even though the precise mechanisms related to venous malformation are still unclear, the clinical manifestations sometimes threaten vital signs such as mastication, airway and phonics. Our therapeutic modalities were reviewed, and their effectiveness and related complications were analyzed. Between March, 1998 and February, 2006, 11 patients (15-59 years old; average 32.4 +/- 13.60, 4 women and 7 men) with craniofacial venous malformation were included in this investigation. All cases experienced some kind of surgery at least once during clinical follow-up. Direct puncture scintigraphy with technetium-99m Sn colloid-labeled demonstrated low-flow malformations in all cases. Two cases underwent bone surgery and another two cases had static suspensions for facial nerve paralysis. Blood loss from surgery alone was 1352 +/- 1115.0 mL, simultaneous procedures yielded 400 +/- 244.9 mL blood loss and sclerotherapy alone resulted in 187 +/- 284.8 mL of blood loss (surgery alone versus sclerotherapy alone, P < 0.01). Excellent sclerotherapy cases were when the malformation was localized and the number of sclerotherapies was significantly fewer than good cases (1.3 +/- 0.58 times versus 3.6 +/- 1.15 times, excellent, good, respectively, P < 0.05). Although there are difficulties in understanding the mechanisms and multiple therapeutic interventions are required, there have been satisfactory outcomes so far and the development of better sclerosants or a real-time navigation system may benefit more precise therapeutic effects and lower morbidity.

Although there are numerous investigations describing bone marrow cells or bone-marrow derived cells at the site of such injuries as bone fractures, infarction and subsequent ischemic reperfusion injury, or cutaneous wounds, little is know about the factors that affect the cells in those clinical situations. Cysteinyl leukotrienes have been extensively investigated in airway diseases that may eventually lead to lung fibrosis; while the engraftment of mesenchymal stem cells have been shown to reverse bleomycin-induced lung fibrosis in vivo. Therefore, we elucidated the involvement of cysteinyl leukotrienes in human mesenchymal stem cell proliferation and differentiation. Human mesenchymal stem cells express the cysteinyl leukotriene type 1 receptor. Various doses of pranlukast, which is a specific cysteinyl leukotriene type 1 receptor antagonist, failed to affect the proliferation of cells; however, 10(-6) M of pranlukast significantly induced cellular cytoplasmic differentiation by showing microvilli sprouting and the emersion of rough endoplasmic reticulum within a 16-hour(s) incubation. Additionally, pranlukast-induced fibronectin protein production by human mesenchymal stem cells. Therefore, attenuation of the cysteinyl leukotriene pathway contributes to human mesenchymal stem cell differentiation and may contribute to modulation of the local injury site.

To avoid hypertrophic scars in burn wounds, the simultaneous application of basic fibroblast growth factor (bFGF) with regular surgical debridement and skin grafting was investigated for skin hardness by clinical examination and instrumental measurement. As little is known about the role of bFGF in wounds, burn wound scars were tested for hardness. Burn scars in various anatomical locations at least 1 year after final wound healing clinically demonstrated a significantly lower hard score in bFGF-treated wounds than in non-bFGF wounds (0.95+/-0.51 versus 2.3+/-0.66, respectively, p

Background Pituitary tumor transforming gene (PTTG), homologous to a mammalian securin, plays a pivotal role in cell transformation, however, its biological function(s) in normal tissues is not fully understood. Because the liver is a regenerative organ, the relevant biological function of PTTG in the liver would be more feasible to understand PTTG. Also, PTTG may be involved in the liver regeneration. Materials and methods Expressions of rat hepatic PTTG messengerRNA (mRNA) and cellular immunoreactivities during the cell proliferative period of the liver regeneration both in vitro and in vivo were tested. Results PTTG expression of the rat primary hepatocyte was stimulated by HGF in a dose dependent manner, and was suppressed when hepatocyte proliferation was inhibited by transforming growth factor-β1. A positive PTTG immunoreactive co-localizing with 5-bromo-2′-deoxyuridine (BrdU) in the hepatocyte nucleus was found and there was a concurrent sister chromatin itself by the immunofluorescent labeling of PTTG with cytokeratin 18 (CK18). Discussion Since the correlation of PTTG mRNA expression, cell proliferation and immunoreactivity were observed in primary rat cultured hepatocytes, PTTG may be a novel marker of cell proliferation both in vitro and in vivo liver regeneration.

Summary Background Large or deteriorated skin defects are sometimes life threatening. There is increasing evidence that adult stem cells are useful for tissue regeneration. Human mesenchymal stem cells (hMSCs) are self-renewing and are potent in differentiating into multiple cells and tissues. Objectives To investigate the effects of hMSCs in cutaneous wound healing. Methods Wound healing was studied in an hMSC-populated porcine skin substitute, using a nude rat model to minimize immune reactions. Full-thickness skin and soft tissue defects of 1AE5 · 1AE5 cm in size, including the panniculus carnosus, were excised and covered with hMSCs and basic fibroblast growth factor (bFGF)soaked skin substitutes and an evaluation was made of wound size, histology and protein expression at 3, 7 and 42 days after injury. Results The wound size was significantly smaller in the hMSC-treated groups (P

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with bone morphogenetic protein-2 alone, or basic fibroblast growth factor alone or combinations of both proteins under serum-free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by bone morphogenetic protein-2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence-activated cell sorting also showed a similar pattern of cell cycle progression with bone morphogenetic protein-2 treatment in serum-free medium and 10% fetal bovine serum treatment. The bone morphogenetic protein-2-treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well-developed granular endoplasmic reticulum, indicative of protein production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by bone morphogenetic protein-2 treatment in serum-free medium.

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivodiffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 μM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1–34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1–34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

To understand the involvement of thyroid hormone on the postnatal development of hypothalamic thermosensitive neurons, we focused on the analysis of thermosensitive neuronal activity in the preoptic and anterior hypothalamic (PO/AH) regions of developing rats with and without hypothyroidism. In euthyroid rats, the distribution of thermosensitive neurons in PO/AH showed that in 3-week-old rats (46 neurons tested), 19.5% were warm-sensitive and 80.5% were nonsensitive. In 5- to 12-week-old euthyroid rats (122 neurons), 33.6% were warm-sensitive and 66.4% were nonsensitive. In 5- to 12-week-old hypothyroid rats (108 neurons), however, 18.5% were warm-sensitive and 81.5% were nonsensitive. Temperature thresholds of warm-sensitive neurons were lower in 12-week-old euthyroid rats (36.4+/-0.2 degrees C, n = 15, p

1. The exact role of the parathyroid hormone-related peptide (PTHrP) is not fully understood. We used immunohistochemistry to localize the PTHrP and its receptor in the brain of the red stingray, particularly in the saccus vasculosus (SV) and choroid plexus. 2. Immunoreactive PTHrP and its receptor were detected in the epithelial cells of the SV and the choroid plexus. In addition, the neuronal perikarya in the nucleus of the SV located in the hypothalamus is positive for the PTHrP. 3. No PTHrP-containing neurons were detected in the choroid plexus. Extracts of SV and choroid plexus showed positive reactions against the PTHrP and its receptor antibody in Western blot analysis. 4. High levels of immunoreactive PTHrP were detected in the plasma equivalent to those present in human humoral malignant hypercalcemia. In contrast, the immunoreactive PTHrP concentration in the cerebrospinal fluid was below detectable levels. 5. Our results suggest that the regulation of the PTHrP in the SV differs from that in the choroid plexus in the red stingray.

Color changes of visible and exposed body surfaces, such as the face and extremities, after burn injury or surgery, such as skin grafting, flap, or sclerotherapy for vascular malformations, are sometimes a concern. The consequences reduce the satisfaction of both patients and physicians. An easy and reproducible method has not yet been established for an objective analysis of color changes; therefore, we tested a hand-held color analyzer (NF-333; Nippon Denshoku Co. Ltd) with data transport to a computer database and analysis software for posttreatment skin color change. The parameters included L, a, and b, which measure clarity, red, and yellow, respectively. Two groups were prospectively divided with 20 (11 females and nine males) patients per group. One group received skin grafting plus basic fibroblast growth factor (bFGF) spray daily and the other group received only skin grafting. The patients were randomized by the date of their first visit to our hospital. Patients were treated with bFGF on odd days, while patients who came on even days were included in the non-bFGF-treated group. The donor site for skin grafting was the lateral thighs and the thickness was similar in both groups. The results were compared at 1-year posttreatment follow-up. Clinical and objective assessments of the scars were performed 1 to years after complete healing. Color change differentials in comparison with the surrounding skin were lower with bFGF treatment in all parameters (p0.01), along with clinical assessment with the Vancouver Scar Scale; therefore, the treatment contribute to a better color match with skin grafting postoperatively.

Second-degree burns are sometimes a concern for shortening patient suffering time as well as the therapeutic choice. Thus, adult second-degree burn patients (average 57.8 +/- 13.9 years old), mainly with deep dermal burns, were included. Patients receiving topical basic fibroblast growth factor (bFGF) or no bFGF were compared for clinical scar extent, passive scar hardness and elasticity using a Cutometer, direct scar hardness using a durometer, and moisture analysis of the stratum corneum at 1 year after complete wound healing. There was significantly faster wound healing with bFGF, as early as 2.2 +/- 0.9 days from the burn injury, compared with non-bFGF use (12.0 +/- 2.2 vs. 15.0 +/- 2.7 days, p0.01). Clinical evaluation of Vancouver scale scores showed significant differences between bFGF-treated and non-bFGF-treated scars (p0.01). Both maximal scar extension and the ratio of scar retraction to maximal scar extension, elasticity, by Cutometer were significantly greater in bFGF-treated scars than non-bFGF-treated scars (0.23 +/- 0.10 vs. 0.14 +/- 0.06 mm, 0.59 +/- 0.20 vs. 0.49 +/- 0.15 mm: scar extension, scar elasticity, bFGF vs. non-bFGF, p0.01). The durometer reading was significantly lower in bFGF-treated scars than in non-bFGF-treated scars (16.2 +/- 3.8 vs. 29.3 +/- 5.1, p0.01). Transepidermal water loss, water content, and corneal thickness were significantly less in bFGF-treated than in non-bFGF-treated scars (p0.01).

Although a number of cytokine or growth factor therapies for wound acceleration have been reported, few mentioned the quality of the outcome. The lower extremity is important in esthetics as well as in function, because it is exposed. Recently, a growth factor, namely basic growth factor (bFGF) is widely used for difficult wound healing with a porcine-derived bilayered artificial dermis for better wound closure. Thus, their combination use was tested clinically.Sequential lower extremity reconstruction by an artificial dermis with or without bFGF administration and secondary split-thickness skin grafting was measured for hardness using a durometer, and the moisture parameters assessed such as effective contact coefficient, transepidermal water loss (TEWL), water content and thickness using a moisture meter for at least 6 months after the final procedure and compared with normal skin control.There was significantly less skin hardness using a durometer in bFGF treatment compared with non-bFGF treatment (16.2 +/- 3.83 vs. 29.2 +/- 4.94, p0.01). Effective contact coefficient, TEWL, water content, and thickness in non-bFGF treatment were all significantly greater than those in bFGF treatment, whereas water content and thickness in bFGF treatment were comparable with those of the control.The use of bFGF as artificial dermis for extensive and deeper tissue loss coverage demonstrated better reconstruction quality in terms of hardness using a durometer and the function of the stratum corneum by moisture analysis.

Pediatric burn wounds can be problematic because an accurate evaluation is difficult as the result of anatomically immature vasculature or immobilization failure, especially in patients with second-degree burns, and because the burn surface areas and the burn depth tend to worsen over the course of time. Delayed wound healing results in unsightly scarring, such as hypertrophic scars, which are problematic both esthetically and functionally. Among cytokines and growth factors, basic fibroblast growth factor (bFGF) is clinically proven, having demonstrated accelerated acute and chronic wound healing. Accelerated wound healing may lead to improved scarring. To elucidate the effects of bFGF on second-degree pediatric burn wounds, a comparative study was performed. A total of 20 pediatric patients ranging from 8 month to 3 years (average 1 year, 3 months +/- 6 months) who suffered from the burns by various causes were divided into two groups, conventional (n = 10) and treatment with bFGF (n = 10). A moisture meter, used to objectively measure the stratum corneum and epithelial-mesenchymal functions, was used to assess scars at least 1 year after wound healing. Clinical evaluation of pigmentation, pliability, height, and vascularity demonstrated significant differences between conventional and bFGF-treated scars (1.7 +/- 0.55 vs 0.7 +/- 0.58, 2.4 +/- 0.82 vs 1.1 +/- 0.69, 1.8 +/- 0.66 vs 0.5 +/- 0.57, 1.9 +/- 0.63 vs 0.8 +/- 0.68; conventional vs bFGF-treated, pigmentation, pliability, height, and vascularity, respectively, P < .01). The effective contact coefficient was significantly greater in conventional wounds than bFGF-treated wounds (14.6 +/- 1.68 % vs 8.7 +/- 2.82 %; conventional vs bFGF, P < .01) and bFGF-treated wounds demonstrated significantly less transepidermal water loss values than conventional treatment (8.3 +/- 1.90 g/m/h vs 5.7 +/- 1.85 g/m/hr; conventional vs bFGF, P < .01). Pediatric burn patients treated with bFGF showed less damaging function of the stratum corneum after healing both in clinical assessment and moisture meter analysis.

To investigate circulating cytokine responsiveness in major burns in association with the systemic stress response system, we tested hypothalamic-pituitary-adrenal (HPA) axis markers in extensive burn cases treated in the Department of Plastic and Reconstructive Surgery at Nagasaki University. The HPA axis is a major stress response system, and the leukemia inhibitory factor (LIF) may be a potent mediator of the HPA axis; therefore, circulating LIF levels in burn patients were studied. Twenty extensively burned patients (burn surface area, >20%), ie, 10 women and 10 men, 37 to 77 years of age (average: 59.1 +/- 12.10 years), were assessed. Circulating LIF, adrenocorticotropic hormone (ACTH), other inflammatory markers, and 24-hour urinary free cortisol excretion levels were investigated. LIF levels were greater in patients who died than in those who survived (186.1 +/- 80.41, 83.5 +/- 64.49 pg/ml, respectively, P < .001) at 36 hours after injury. ACTH levels were more significantly elevated in fatal cases than in those who survived. (41.3 +/- 8.28, 25.2 +/- 7.84 pg/ml, respectively, P < .0001). Twenty-four hour (24 to 48 hours after injury) pooled urinary free cortisol excretion levels also were significantly greater in fatal cases than in the surviving patient group (235.0 +/- 36.49 microg/day, 69.0 +/- 18.04 microg/day, respectively, P < .0001). The correlation between serum LIF and urine free cortisol was significant (r = .30; P < .01) as was the correlation of serum LIF with plasma ACTH (r = .24; P < .01). Serum LIF as well as HPA axis activity markers is a good marker of disease severity and prognosis in patients with extensive burns.

Cell-to-cell interactions between human mesenchymal stem cells and potential adjacent cells such as endothelial cells, dermal fibroblasts, and epidermal keratinocytes was investigated. A modified dual Boyden chamber assay using 8-microm pores revealed a more powerful chemotactic cell migration of human mesenchymal stem cells toward human epidermal keratinocytes than other cells, such as umbilical artery endothelial cells and dermal fibroblasts, during 16 hours of incubation (336.2+/-52.33, 36.0+/-11.20, and 62.7+/-18.16, cells/field, respectively, p

In vivo studies using bone marrow-derived mesenchymal stem cells are still uncommon. Applications for bone defect replacement in undesirable clinical circumstances such as large defects, bacterial or other pathogen-contaminated fields, and irradiated surgical wound bed necessitate vascularized bone regeneration. Use of a fascial flap including regenerated bone would be a very powerful tool for treatment. It would be especially beneficial in cases where normal bone regeneration is not expected due to a lack of sufficient blood supply, extensive surgical scarring, or bacterial contamination. In this study, we used nude rats in which the superficial epigastric flap of the experimental group was used to wrap around a mixture of human mesenchymal stem cells, bone morphogenetic protein-2, and basic fibroblast growth factor cytokines in a gelatin carrier. These rats showed significantly higher bone mineral density at 4 weeks compared to the other experimental groups containing phosphate buffered saline, human mesenchymal stem cells alone, or the two cytokines alone (p < 0.01). There were no remarkable histologic differences up to 7 days. At 2 weeks, more progressive vascularity and perivascular tissue deposits were seen in the experimental group. Basophilic mineral structure surrounded the fibroblast-like mesenchymal stem cells at 4 weeks, presumably osteoblastic or osteoclastic cell lining. Bone marker immunohistochemistry against alkaline phosphatase and osteocalcin revealed diffuse and distinct immunoreactivity in osteoblastic cells in the experimental group at 4 weeks. Further transcriptional expression of polyomavirus enhancer binding protein 2alphaA suggested that the human transplanted cells proceeded to osteogenic lineage in 4 weeks. These results may be useful as a new approach for bone regeneration.

Pituitary tumor transforming gene (PTTG), homologous to a mammalian securin, plays a pivotal role in cell transformation, however, its biological function(s) in normal tissues is not fully understood. Because the liver is a regenerative organ, the relevant biological function of PTTG in the liver would be more feasible to understand PTTG. Also, PTTG may be involved in the liver regeneration.Expressions of rat hepatic PTTG messengerRNA (mRNA) and cellular immunoreactivities during the cell proliferative period of the liver regeneration both in vitro and in vivo were tested.PTTG expression of the rat primary hepatocyte was stimulated by HGF in a dose dependent manner, and was suppressed when hepatocyte proliferation was inhibited by transforming growth factor-beta1. A positive PTTG immunoreactive co-localizing with 5-bromo-2'-deoxyuridine (BrdU) in the hepatocyte nucleus was found and there was a concurrent sister chromatin itself by the immunofluorescent labeling of PTTG with cytokeratin 18 (CK18).Since the correlation of PTTG mRNA expression, cell proliferation and immunoreactivity were observed in primary rat cultured hepatocytes, PTTG may be a novel marker of cell proliferation both in vitro and in vivo liver regeneration.

Background and objective: The combined application of cytokines on embryonic fibroblasts and dermal substitute were studied for optimal skin defect coverage. The mechanism of combined treatment of leukemia inhibitory factor (LIF)-transfected embryonic fibroblasts and vascular endothelial growth factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute. Methods: Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full-length LIF cDNA and added to various doses of VEGF for detection of signaling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice up to for 14 days. Results: LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 ng VEGF on days 3 and 5. Erk mitogen-activated protein (MAP) kinase phosphorylation was observed from 1 to 30 min in LIF-transfected and 10 ng of VEFG, and 1 to 60 min in LIF-transfected and 100 ng VEFG treatments. The cellular fibronectin levels also increased in LIF-transfected cells with 10 and 100 ng VEGF additions. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 μg of VEGF markedly enhanced collagen I expression and CD34 angiogenic marker on days 7 and 14. Conclusion: LIF transfection and VEGF treatment enhanced phosphorylated-Erk-MAP kinase in vitro. In vivo study revealed that the combined application of LIF transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of a dermal substitute induced greater skin collagen production and angiogenesis in the dermal substitute.

The quantity of powder food consumed by individual mice was gauged with a newly developed apparatus that includes a specialized feeding station, an electric scale, and an interface to a computer that records the weight of the powder food jar. Using the measurements that exceeded the cutoff value, that is, the threshold between a mouse feeding or drinking event and scale noise, the reconstructed data were presented as the daily pattern of feeding and drinking in time resolution of 9 to 30 min. In this system, the ratio of noise to total consumption value was less than 4%. The fractal structure and fitting curve of this time series data were also analyzed by the nonlinear least-squares method, combined with the maximum entropy method. These analyses demonstrated that the mouse feeding event has circadian and ultradian periodicity. This apparatus and system are useful tools in studying the daily feeding pattern of mice.

Growth hormone (GH) exerts potent effects on bone metabolism, resulting in an increased bone formation in animals and humans. Acromegaly has been associated with increased bone turnover, whereas the net effect of the increased bone metabolism has been obscured because patients with acromegaly are often associated with hypogonadism. We investigated changes in cortical and cancellous bone in adult rats implanted mammosomatotrophic pituitary tumor cells (GH3) as a model of acromegaly with gonadal dysfunction. Acromegaly model rats were prepared by implanting GH3 cells into female Wistar-Furth rats at 17 weeks of age. At 28 weeks of age, GH3-bearing rats (GH rats) showed very high serum GH levels and a moderate increase in serum prolactin levels, resulting in low circulating estradiol levels. The GH rats showed significant increases in body weight and in length and volume of both the femur and vertebral body. Bone mineral content values of either the midfemur or the whole lumbar body were significantly greater in the GH rats compared with littermate controls, while the areal bone mineral density values of the respective bones were not different between the two groups. The parameters of mechanical strength of the femur were significantly larger in the GH rats than in controls, whereas those of the lumbar vertebral body cylinder specimen were not different between the two groups. Respective normalized mechanical parameters of the femur and the vertebral body were the same in the GH rats as in controls. In the midfemur, the GH rats showed a significant increase in the total cross-sectional area without influencing the bone marrow area, resulting in an increase in the cortical bone area and the moment of inertia compared with controls. The indices of periosteal bone formation in the midfemur were greater in the GH rats compared with controls, but the endocortical bone formation and resorption were not different between the two groups. In the vertebral body cancellous bone, the GH rats had an increase in bone turnover rate, whereas the structural parameters were not different between the two groups. These results from GH3-bearing rats demonstrate that an excess of GH increases cortical bone mass in rats accompanied with estrogen deficiency, while no large effect on vertebral body cancellous bone mass is seen.

Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.

The basic fibroblast growth factor (bFGF) is known to proliferate and maintain the adult stem cells. The human mesenchymal stem cells (hMSCs) are very homogenous and constitute of euchromatin nuclei in 10% fetal bovine serum (FBS) and induce the heterochomatin nuclei by bone morphogenetic protein. In order to further investigate other growth factor, other cell types like bFGF, keratinocytes, dermal fibroblasts and endothelial cells, the cellular and molecular analyses are performed in vitro assay systems. The various doses of bFGF (0.25–25 μg/ml) significantly proliferate the hMSCs in time-dependent manner in s serum-free medium. The ultra-structure by electron microscopy revealed the very small, round and immature both cytoplasmic and nuclear structure resembling the 10% FBS-cultured cells. The hMSCs and bFGF and other cells are co-cultured in 8 μm-pore chamber systems for 16-hour incubation. The hMSCs successfully migrate through the pore when keratinocytes, dermal fibroblasts, endothelial cells or bFGF (345.0 ± 68.86, 63.8 ± 16.50, 36.2 ± 9.60, 12.0 ± 4.89, p

Skin and soft tissue defects are sometimes problematic especially when the defects large, contaminated, irradiated, or poor blood supplied. The human mesenchymal stem cells (hMSCs) are proliferated upon basic fibroblast growth factor (bFGF) stimuli in vitro and in vivo. In this experiment, the skin and soft tissue defects are investigated if the wounds are able to be reepithelialized or accelerated by hMSCs, bFGF and porcine-derived bilayered skin template. 1.5 × 1.5 cm2 nude rat skin and soft tissue defects including panniculus carnosus are excised and 1 × 106 hMSCs and various doses of bFGF (1–100 μg) applied. Before and after normal reepithelialization, the tissues are tested for protein expressions by immunohistochemistry and Western blotting. The wound sizes are significantly decreased at day 7 with hMSCs with 1, 10, or 100 μg bFGF compared to hMSCs-alone or medium-only. All the wounds healed by day 42. 42 Kda and 38 Kda human-derived pancytokeratin expressions, which do not cross-react with murine antigens, by Western blotting significantly augmented by 10 μg bFGF compared to hMSCs-alone. The epidermal immunolocalizations such as integrin α3 and SKALP (Skin-derived Anti Leukoproteinase) are greatly elevated in time and dose-dependent manner. Human pan-cytokeratin expressions are immunoreactive even at day 42. These data suggest the skin and soft tissue wound healing is accelerated by hMSCs together with bFGF, partly by means of differentiation of hMSCs toward epidermal components.

Human mesenchymal stem cells (hMSCs) obtained from a single donor from an iliac crest, were investigated with cutaneous wound healing models using nude rat, eliminating T cell-mediated immune reaction to the grafted stem cell of human origin. Co-culture of the human keratinocytes and the hMSCs revealed tight basal membranous interaction by electron microscopy. 1.5 × 1.5 cm2 dorsal full-thickness defects including panniculus carnosus of F344/NJCl-rnu nude rats were covered by bi-layered porcine-derived collagen sponge artificial skin substitutes, Pelnac®, Johnson & Johnson, Tokyo, Japan, impregnated with 5 × 106 hMSCs grated to along with 0, 1, 10, or 100 μg of recombinant human basic fibroblast growth factor (bFGF). The tissues were harvested at 3, 7 42 days after grafting. The defects were remarkably epithelialized by 7 days after coverage with artificial skin substitutes and 10 μg of bFGF, while artificial substitutes with no hMSCs or artificial skin substitutes with hMSCs alone did not demonstrate such healing. The architectures of the artificial skin substitutes were integrated by day 42 in hMSCs-treated groups. The artificial skin substitute at least plays a role in as a template or scaffold of the grafted hMSCs. Up to day 3, the mesenchymal stem cell surface markers such as CD 29 and CD 44 were remained immunopositive in the groups with hMSCs-treated groups over the reconstructed dermis-like tissue. By 42 days after grafting, artificial skin substitutes with hMSCs and 10 μg of bFGF demonstrated the total epithelization and the keratinocytes by this treatment exhibited the pan-cytokeratin of human origin by immunohistochemical expressions. Thus, the hMSCs with bFGF treatment using an artificial skin substitute as a successfully heal. These results suggest that the human mesenchymal stem cells may be utilized for wound coverage together with bFGF and artificial skin substitutes.

The combined application of cytokines on embryonic fibroblasts and dermal substitute were extensively studied for optimal skin defect coverage. Signalling of combined treatment of leukemia inhibitory factor (LIF) and vascular endothelial factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute. Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full length LIF cDNA and added to various doses of VEGF for detection of signalling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice for 14 days. LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 nM VEGF on days 3 and 5. LIF-transfected cells showed rapid phosphorylation of STAT 3 from 1 minute to 60 minutes after VEGF treatment, while vector-transfected cells failed to induce such phosphorylation after VEGF treatment. Erk MAP kinase phosphorylation was observed from 1 to 15 minutes in LIF-transfected and 10 nM of VEFG and 1 to 30 minutes in LIF-transfected and 100 nM VEFG treatments. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 μg of VEGF markedly enhanced collagen I expression and CD 34 angiogenic marker on days 7 and 14. LIF transfection induced constitutive STAT signaling and enhanced phosphorylated-Erk MAP kinase with exogenous VEGF. In vivo study revealed that the combined application of LIF-transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of an artificial dermis.

Critically larger bone and skin defects often lack the enough nutrient blood supply to induce normal wound healing. Additionally, deteriorated environment such as poor vascularity due to the hard fibrosis after irradiation or extensive tissue loss due to the highly damaged tissue or severe bacterial contamination, leads to finding another donor sites or another methods to repair. Potential free vascularized flaps are widely accepted for reconstruction purpose, however, the donor-site morbidity is sometimes concerned and its clinical application may be limited. Tissue engineered tissues are now most actively investigated and partially clinically appropriate for use. In order to assist the bone wound healing, the human mesenchymal stem cells (hMSCs), wrapped with the abdominal fascial flap, which is nutrient of superficial epigastric vascular systems in bone, and the full-thickness dorsal defect covered by artificial skin substitute as a carrier in skin, are used as cell source with potential differentiation with specific cytokines in a nude rat model for eliminating T cell immunity. The hMSCs and osteogenic cytokines such as bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) were intra-arteriallly injected and incubated for 10 minutes and then the gelatin-carrier were wrapped with the abdominal superficial fascia and investigated for the subsequent experiments. The heterotopic bone formation was most significantly observed in 4 weeks after injection and this may be used clinically to enhance the compromised bone healing.

Vascular leiomyomas are believed to arise from the smooth muscle of blood vessels and are characterized by the proliferation of vascular smooth muscle cells (VSMC) and numerous slit-like vascular lumens. Parathyroid hormone (PTH)-related peptide (PTHrP) plays an important role in local autocrine and/or paracrine regulation of cellular growth and function in VSMC. To investigate the interaction between VSMC and endothelial cells, we evaluated the distribution of PTHrP and PTH/PTHrP-receptor in 10 vascular leiomyomas of the skin by immunohistochemistry and in situ hybridization (ISH) of paraffin-embedded specimens. Both immunohistochemistry and ISH revealed that PTH/PTHrP-receptors are expressed in endothelial cells lining areas with slit-like vascular lumens and very weakly expressed in proliferating VSMC in all vascular leiomyomas. On the other hand, PTHrP itself was localized mainly in proliferating VSMC. These results support the hypothesis that PTHrP acts through the PTH/PTHrP-receptor via an autocrine and/or paracrine mechanism from VSMC to endothelial cells in the formation of characteristic microenvironments of vascular leiomyoma cell composition.

Keloid is a dermal fibroproliferative tissue of unknown etiology. Protein tyrosine kinases (PTKs) play an important role in the regulation of cell growth and differentiation. Activation of PTK cascades in keloid fibroblasts is thought to be closely linked to abnormal cell proliferation and migration. We determined the expression profile of PTK genes in normal skin and keloid fibroblasts using the homology cloning method with a degenerated primer. Eight PTK genes were expressed among a total of 46 receptor-type clones. The most abundant type of PTK receptors was the platelet-derived growth factor receptor in both fibroblasts. However, insulin-like growth factor-I receptor (IGF-IR) was overexpressed only in keloid-derived fibroblasts (9 of 24). Immunohistochemical analysis confirmed the high expression of IGF-IR in keloid fibroblasts, but not in normal fibroblasts. To examine the functional properties of the IGF-I/IGF-IR pathway, we investigated cell proliferation and invasion activities of both types of fibroblasts. The mitogenic effect of IGF-I on both fibroblasts was very weak compared with serum stimulation. In contrast, the invasive activity of keloid fibroblasts was markedly increased in the presence of IGF-I, and inhibited by a neutralizing antibody against IGF-IR. Our results indicate the involvement of activated IGF-I/IGF-IR in the pathogenesis of keloid by enhancing the invasive activity of fibroblasts.