Prognostic nutritional index and early mortality with percutaneous endoscopic gastrostomy.

BACKGROUND: Although percutaneous endoscopic gastrostomy (PEG) is a well-accepted and less invasive method of feeding tube placement in patients with swallowing difficulties, complications and early death after PEG have been reported. AIM: This study aimed to evaluate predictive factors associated with 30-day mortality after PEG, and to assess the utility of nutritional supporting period before PEG in reducing early mortality following PEG. DESIGN: An observational study. METHODS: We retrospectively analyzed 268 patients who underwent PEG at Sapporo Shirakaba-dai Hospital from 2006 to 2010, using clinical and laboratory data to analyze predictive factors associated with early death after PEG. Then, we prospectively assessed 152 consecutive patients assessed for eligibility for PEG from 2011 to 2014. We assessed the patients' nutritional condition using Onodera's prognostic nutritional index (PNI), and supported nutrition for more than 10 days before PEG in patients with a poor nutritional index (PNI < 37). RESULTS: In both univariate and multivariate analyses in the retrospective study, Onodera's PNI of less than 37 was the only predictive factor for early mortality. In the second study, among the 115 patients who finally underwent PEG, early mortality rates improved to 1.7% from 5.2% in the first study. Conversely, 32% of patients with malnutrition who did not undergo PEG died within 30 days. CONCLUSION: Nutritional status might be a predictive factor for early mortality after PEG. In patients with poor nutritional status, nutritional supporting period before PEG might improve the outcomes and reduce unnecessary PEG.

Relationship between folic acid intake and gingival health in non-smoking adults in Japan.

OBJECTIVE: To assess the relationship between dietary intake of folate and gingival bleeding in non-smoking adults in Japan. MATERIALS AND METHODS: Data were obtained from residents who participated in the regional nutrition survey and survey of dental diseases conducted by the administrative office of northernmost prefecture of Japan. Dietitians visited households to collect data on dietary intake. Clinical parameters, including Community Periodontal Index (CPI) and bleeding on probing (BOP), were examined in community centers. Information on smoking habit was obtained from the interview. Then the data from 497 non-smoking adults with 20 teeth or more, aged 18 years or older, were analyzed. The relationship between dietary intake of folic acid and gingival bleeding status was estimated using multivariate analysis. RESULTS: Pearson's correlation coefficient showed a significant negative correlation between dietary folate level and bleeding on probing. The negative association between folate level and bleeding on probing remained statistically significant in multiple regression analysis (standardized beta = -0.204, P < 0.001). However, no significant association was found between CPI scores and folate intake level. CONCLUSIONS: The results suggest that dietary intake of folic acid, an important indicator of gingival bleeding in adults, may provide an important clinical target for intervention to promote gingival health.

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines.

Transcription factor 2 gene (TCF2) encodes hepatocyte nuclear factor 1beta (HNF1beta), a transcription factor associated with development and metabolism. Mutation of TCF2 has been observed in renal cell cancer, and by screening aberrantly methylated genes, we have now identified TCF2 as a target for epigenetic inactivation in ovarian cancer. TCF2 was methylated in 53% of ovarian cancer cell lines and 26% of primary ovarian cancers, resulting in loss of the gene's expression. TCF2 expression was restored by treating cells with a methyltransferase inhibitor, 5-aza-2'deoxycitidine (5-aza-dC). In addition, chromatin immunoprecipitation showed deacetylation of histone H3 in methylated cells and, when combined with 5-aza-dC, the histone deacetylase inhibitor trichostatin A synergistically induced TCF2 expression. Epigenetic inactivation of TCF2 was also seen in colorectal, gastric and pancreatic cell lines, suggesting general involvement of epigenetic inactivation of TCF2 in tumorigenesis. Restoration of TCF2 expression induced expression of HNF4alpha, a transcriptional target of HNF1beta, indicating that epigenetic silencing of TCF2 leads to alteration of the hepatocyte nuclear factor network in tumours. These results suggest that TCF2 is involved in the development of ovarian cancers and may represent a useful target for their detection and treatment.

Human mesenchymal stem cells successfully improve skin-substitute wound healing.

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 1.5 x 1.5 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 < 0.01) and any dose of bFGF (1, 10, 100 microg) enhanced the healing (P < 0.01). The re-epithelialization markers integrin alpha3 and skin-derived antileucoproteinase were remarkably increased with the presence of bFGF in a dose-dependent manner, while the mesenchymal cell surface markers CD29 and CD44 were downregulated in a time-dependent manner. Human pancytokeratin, which does not cross-react with rat antigens, was observed by Western blotting at 38 kDa and 42 kDa from the hMSC-treated tissues on day 7. The expression levels were elevated by 10 microg bFGF (P < 0.01). The immunohistochemical expression of human pancytokeratin was only observed in the hMSC-treated groups. CONCLUSIONS: These data suggest that hMSCs together with bFGF in a skin defect model accelerate cutaneous wound healing as the hMSCs transdifferentiate into the epithelium.

Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours.

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2'-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5' region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5' CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours.

Parathyroid hormone-related peptide is a potent tumor angiogenic factor.

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 vivo diffusion 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 microM), 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.

A new apparatus for studying feeding and drinking in the mouse.

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.

Octreotide treatment suppresses malignant somatotrophic pituitary tumor cell growth in rats.

We have recently established an in vitro cell line (metastatic mGH3) derived from lymph node metastases of the rat pituitary somatotroph. Here we examined the in vivo effects of octreotide, a somatostatin analog, against malignant pituitary tumors. Wistar-Furth rats (n=8) were inoculated subcutaneously with mGH3 cells while control rats received injections of equal volumes of the vehicle only. Four rats were treated with octreotide three times daily while another group of four rats were treated with saline only. After 6 weeks of treatment, histopathological and immunohistological analyses were performed. The tumor weights of rats treated with octreotide were significantly lighter than those of untreated rats. All rats implanted mGH3, but not administered treatment, developed inguinal lymph node metastases, whereas none of those implanted mGH3 and treated with octreotide developed such metastases. The proportion of PCNA-stained tumor cells was higher in tumors of untreated rats than in those of octreotide-treated rats. However, the proportion of apoptotic cells in the tumor was not different between treated and untreated rats. Our results suggest that octreotide might be potentially effective for invasive and malignant human pituitary tumors by regulating the tumor cell cycle.

Skeletal changes in rats bearing mammosomatotrophic pituitary tumors: a model of acromegaly with gonadal dysfunction.

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.

Leukemia inhibitory factor enhances bone formation in calvarial bone defect.

For bone defect reconstruction, locally administered cytokine plasmid was examined. Leukemia inhibitory factor (LIF) can bind to the osteoblast cell surface and induce bone formation both in vitro and in vivo. The authors investigated the local mouse LIF complementary deoxyribonucleic acid (cDNA) plasmid in the pcDNA 3 expression vector, which is promoted by cytomegalovirus and is stabilized by bovine growth hormone polyadenylation, with a gelatin sponge carrier. A total of 150 male Wistar rats were used. They were divided into three groups. Group 1 (N = 30) was treated with the gelatin carrier of the pcDNA 3 vector, group 2 (N = 90) was treated with three different doses of LIF cDNA (0.1, 1, and 10 micrograms) in the pcDNA 3-vector plasmid along with the gelatin carrier, and group 3 (N = 30) was treated with recombinant human bone morphogenetic protein -2. Ten animals in each group were euthanized at 1, 3, and 5 weeks postoperatively. Animals treated with LIF cDNA showed significantly enhanced bone mineral density (p < 0.05), as confirmed by dual-energy X-ray absorptiometry (DEXA), in 3 weeks compared with the control vehicle. By 3 weeks, the number of fibroblast-like cells and collagen fibers decreased, whereas the osteoblast-like cells increased inversely, as revealed during histological examination. LIF messenger ribonucleic acid demonstrated by in situ hybridization was observed most markedly in osteocytes of the LIF cDNA-treated group. Also, LIF peptide was detected in the same cell type by immunohistochemistry. Locally administered LIF cDNA plasmid in a gelatin carrier can increase bone density significantly, with subsequent bone formation, probably via osteocyte activation.

Expression of insulin-like growth factor I messenger ribonucleic acid in developing osteophytes in murine experimental osteoarthritis and in rats inoculated with growth hormone-secreting tumor.

Osteophytes are one of the characteristic features of osteoarthritis and are often found in acromegalic arthropathy. The aim of this study was to investigate insulin-like growth factor I (IGF-I) involvement in osteophyte formation. One percent collagenase solution was injected into murine knee joints as an osteoarthritis model. In a different animal group, GH-secreting tumor cells were inoculated s.c. to the rat thigh as an acromegaly model. A series of osteophyte formation was examined histologically. IGF-I messenger RNA was detected using the in situ hybridization method. Type I IGF receptors were detected immunohistochemically. In the osteoarthritis model, osteophyte formation appeared as synovial or perichondral cell proliferation adjacent to the articular cartilage on day 5, followed by cartilage formation on day 7 and endochondral ossification on day 14. In the acromegaly model, synovial or perichondral cell proliferation was observed 4 weeks after inoculation, followed by osteophyte formation at 8 weeks. In both models, IGF-I messenger RNA and type I IGF receptor were coexpressed by proliferating synovial or perichondral cells, proliferating chondrocytes, and osteoblasts within the developing osteophytes. These results suggest that IGF-I regulated the initiation and development of osteophyte formation in both models in an autocrine and/or paracrine fashion.

Developmental and hormonal regulation of thermosensitive neuron potential activity in rat brain.

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<0.01,) than in 3-week-old and in 5-week-old euthyroid rats (38.5+/-0.5 degrees C, n = 9 and 38.0+/-0.3 degrees C, n = 15, respectively). The temperature thresholds of warm-sensitive neurons in 12-week-old hypothyroid rats (39.5+/-0.3 degrees C, n = 8) were similar to that of warm-sensitive neurons of 3-week-old raats (euthyroid and hypothyroid). In contrast, there was no difference in the thresholds of warm-sensitive neurons between hypothyroid and euthyroid rats at the age of 3-5 weeks. In conclusion, monitoring the thermosensitive neuronal tissue activity demonstrated the evidence that thyroid hormone regulates the maturation of warm-sensitive hypothalamic neurons in developing rat brain by electrophysiological analysis.

Overexpression of insulin-like growth factor-1 (IGF-I) receptor and the invasiveness of cultured keloid fibroblasts.

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.

Therapeutic usefulness of wild-type p53 gene introduction in a p53-null anaplastic thyroid carcinoma cell line.

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.

Distribution of the parathyroid hormone-related peptide and its receptor in the saccus vasculosus and choroid plexus in the red stingray (Dasyatis akajei: Elasmobranch).

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.

Antisense inhibition of parathyroid hormone-related peptide gene expression reduces malignant pituitary tumor progression and metastases in the rat.

A newly established metastatic rat pituitary tumor (mGH3) possesses a malignant phenotype that is invasive and hypervascular compared with the original GH3 tumors. mGH3 cells exhibit anchorage independence and expression of elevated levels of parathyroid hormone-related peptide (PTHrP) in vitro. To clarify the role of PTHrP in the development of the malignant phenotype, tumor cells were treated with phosphorothioate antisense PTHrP oligonucleotide. Treatment with antisense PTHrP resulted in a scattering phenomenon in the colony formation assay but did not inhibit cell growth in vitro. Inoculation of mGH3 cells in the cerebral ventricle resulted in a rapid growth of tumor cells within 3 weeks and dissemination throughout the entire ventricular system. Although treatment with sense or mismatched PTHrP oligonucleotide did not influence the subsequent tumor growth, the in vivo coinjection and injection of antisense PTHrP 1 week after tumor cell implantation into the right lateral ventricle markedly reduced tumor size and suppressed metastasis formation. The survival rate of mGH3 tumor-injected rats was prolonged by antisense PTHrP therapy. Our results demonstrated the biological involvement of PTHrP in malignant phenotype in rat pituitary tumors, suggesting that antisense PTHrP may provide a novel antimetastatic therapy for malignant somatotroph tumors.

Parathyroid hormone-related peptide expression in endocrine tumors.

Parathyroid hormone-related peptide (PTHrP) expression is associated with the histological type in pituitary tumor, but not in thyroid carcinoma. However invasive and metastatic tumors showed intense PTHrP staining in both pituitary and thyroid tumors. Analysis of the established metastatic rat pituitary tumor cell line showed that PTHrP plays a crucial role in in vivo cell proliferation closely related to worsening of malignant transformation and neovascularization in a paracrine fashion.