Interaction of hydrophobic components in female urine before and after childbirth with P-glycoprotein in vitro.

The first urine in the morning (total 15 samples) and whole day urine (total 4 days, 17 samples) were collected from a young healthy woman during the pregnancy and lactation period, to examine the possible interactions of urine components (methanol extracts) with P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs). The interaction was evaluated by measuring the intracellular accumulation of rhodamine123, a P-gp substrate, in LLC-GA5-COL150 cells, or calcein, an MRP substrate, in Caco-2 cells in the absence and presence of urine components. Four first urine samples out of 12 collected before childbirth and one sample out of three collected after childbirth suppressed P-gp function significantly. The effect of pregnancy and lactation on P-gp inhibitory potencies of urine components was not observed. The whole day urine samples showed a clear circadian rhythm, in which three first urine samples in the morning out of four showed greater P-gp inhibitory potencies than other daytime samples. Interaction of urine components with MRPs was not detected. In conclusion, the concentration of endogenous P-gp inhibitor(s) was higher in the first urine in the morning, showing a clear circadian rhythm. Normal pregnancy and lactation appeared not to significantly affect the P-gp inhibitory potencies of urine components.

Risk factors for major morbidity after liver resection for hepatocellular carcinoma.

BACKGROUND: Bile leakage, and organ and/or space surgical-site infection (SSI) are common causes of major morbidity after partial hepatectomy for hepatocellular carcinoma (HCC). The purpose of this study was to analyse risk factors for major morbidity and to explore strategies for its reduction after partial hepatectomy for HCC. METHODS: Risk factors for bile leakage and organ/space SSI were analysed in patients who underwent partial hepatectomy for HCC between 2001 and 2010. The causes, management and outcomes of intractable bile leakage requiring endoscopic therapy or percutaneous transhepatic biliary drainage were analysed. In addition, causative bacteria, outcomes and characteristics of organ/space SSI were investigated. Risk factors were identified using multivariable analysis. RESULTS: Some 359 patients were included in the analysis. The prevalence of bile leakage and organ/space SSI was 12·8 and 8·6 per cent respectively. Repeat hepatectomy and an operating time of at least 300 min were identified as independent risk factors for bile leakage. The main causes of intractable bile leakage were latent strictures of the biliary system caused by previous treatments for HCC and intraoperative injury of the hepatic duct during repeat hepatectomy. Independent risk factors for organ/space SSI were repeat hepatectomy and bile leakage. Methicillin-resistant Staphylococcus aureus was detected more frequently in organ/space SSI after repeat hepatectomy than after initial partial hepatectomy. CONCLUSION: Repeat hepatectomy and prolonged surgery were identified as risk factors for bile leakage after liver resection for HCC. Bile leakage and repeat hepatectomy increased the risk of organ/space SSI.

Recognition of hand disinfection by an alcohol-containing gel using two-dimensional imaging in a clinical setting.

BACKGROUND: Hand hygiene compliance is important for the prevention of healthcare-associated infections. The conventional method of measuring hand disinfection guidelines involves an external observer watching the staff personnel, which introduces bias, and observations are only made for a set period of time. An unbiased, non-invasive automated system for assessing hand sanitization actions can provide a better estimate of compliance. AIM: To develop an automated detector to assess hand hygiene compliance in hospitals, without bias from an external observer, capable of making observations at different times of the day, as non-invasive as possible by using only one camera, and collecting as much information as possible from two-dimensional video footage. METHODS: Video footage with annotations from various sources was collected to determine when staff performed hand disinfection with gel-based alcohol. The frequency response of wrist movement was used to train a support vector machine to identify hand sanitization events. FINDINGS: This system detected sanitization events with an accuracy of 75.18%, a precision of 72.89%, and a recall of 80.91%. These metrics provide an overall estimate of hand sanitization compliance without bias due to the presence of an external observer while collecting data over time. CONCLUSION: Investigation of these systems is important because they are not constrained by time-limited observations, are non-invasive, and they eliminate observer bias. Although there is room for improvement, the proposed system provides a fair assessment of compliance that the hospital can use as a reference to take appropriate action.

[Relict microorganisms of cryolithozone as possible objects of gerontology].

Permafrost is widely distributed in the Northern Hemisphere, and its age reaches hundreds of thousands and millions of years. Permafrost contains alive microorganisms which are not frozen due to relatively high temperature of the environment (-2...-8 degrees C), but the microorganisms are immobilized and therefore aged probably similar to the age of permafrost. Longevity of the relict microbial cells is related obviously to their mechanism of protection against heat, radiation, free radicals and other damaging agents. A strain of Bacillus sp. was isolated from permafrost aged of about 3 million years, 16S rDNA sequence was identified and preliminary testing of bacterial culture on Drosophila melanogaster and mice was made. Immune stimulation and improvement of physical condition were observed, and that, together with the age of the microbial cells, presents the relict microorganisms as objects of gerontology.

Somatostatin concentration responds to arginine in portal plasma: effects of fasting, streptozotocin diabetes, and insulin administration in diabetic rats.

Changes of somatostatin concentration in response to a single i.v. injection of arginine (400 mg/kg body weight) were examined in extracted portal plasma of normal and diabetic rats in the fully fed state and after 24 h or fasting, as well as in diabetic rats treated with insulin for one week. In both normal and diabetic animals fasted for 24 h, the basal level of somatostatin declined but the magnitude of the arginine-induced elevation of somatostatin was not affected, suggesting a physiologic role of the tetradecapeptide in nutrient homeostasis. When compared with intact rats, diabetic animals were shown to have increased levels of somatostatin before and after arginine administration, both of which were attenuated by insulin replacement therapy. These findings suggest that alterations of D cell function in streptozotocin diabetes may be related to either insulin deficiency or its metabolic consequences.

Physiologic role of somatostatin. Insulin release from rat islets treated by somatostatin antiserum.

In order to clarify the physiologic role of somatostatin in insulin release, rat pancreatic islets treated by somatostatin antiserum were incubated in media containing various concentrations of glucose. Insulin release from antiserum-treated islets was significantly elevated above that from nontreated ones at 3.3 and 8.3 mM glucose, while the former was not different from the latter at 16.7 mM glucose. It is suggested that somatostatin plays an important role in the regulation of insulin release in the physiologic range of glucose concentration.

Reconstitution of peripheral blood lymphocyte subsets in the long-term disease-free survivors of patients with acute myeloblastic leukemia.

The number of long-term survivors of patients with acute myeloblastic leukemia (AML) has increased as a result of the progress of chemotherapy. We examined the recovery of peripheral blood lymphocytes (PBL) subset after chemotherapy to clarify the reconstitution of the immune system in AML. Thirty patients with AML in complete remission (CR) were entered into the study. There were 12 males and 18 females; one M0, six M1, 14 M2, three M3, two M4 and four M5 according to FAB classification. The age ranged from 21 to 78 years (median age, 46 years) and the duration of disease-free survival after completion of chemotherapy ranged from 5 to 122 months (median, 35 months). The chemotherapy was performed according to the protocol designed by the Japan Adult Leukemia Study Group (JALSG). PBL subsets were analyzed by flow cytometry with the use of monoclonal antibodies against CD2, CD3, CD4, CD5, CD8, CD16, CD20, CD45RA, CD56, CD57 and HLA-DR. There was a significant positive relationship between the absolute number of CD4+, CD45RA+ CD4+ cells and the duration of time post-therapy and a significant negative relationship between %CD5+ B, CD56+ cells and the duration of time post-therapy. The appearance of autoantibodies (rheumatoid factor and anti-DNA antibody) tended to increase after 2 years, however, there was no relationship between CD5+ B cells and the frequency of rheumatoid factor. These findings demonstrate that patients in CR have a low number of CD4+ and CD45RA+ CD4+ T cells at an early period after chemotherapy and that each subset recovered to a normal level in 2 years. %CD5+ B and CD56+ cells gradually decreased and returned to their normal level after 4 years. There were high numbers of DR+ T cells and NK cells for a long time, suggesting that activated T cells and NK cells may play a role in the immune surveillance system after chemotherapy.

Origins of hemopoietic and stromal cells in subcutaneous femur implants.

The origin of spleen colony-forming units (CFUs) and fibroblastoid colony-forming cells (CFUF) repopulating the marrow of femurs subcutaneously implanted in syngeneic and semi-syngeneic mice was studied by making use of a chromosome marker for CFUs and a cytotoxicity test for CFUF. The CFUs present in implants during first 4 weeks were of a mixture of those from the donor and the host, and thereafter, were exclusively of host origin. On the other hand, the colony growth of CFUF from the marrow of C57BL/6 femurs grafted in B6CBAF1 mice remained almost totally unaffected by treatment with C57BL/6 anti-B6CBAF1 serum as late as 40 weeks after implantation. Quite similar results were obtained from the marrow-depleted femur implants. It is concluded from the present studies that chimeric marrow consisting of hemopoietic cells of host origin and stromal cells of donor origin can repopulate the femurs within a matter of several weeks after subcutaneous implantation.

Age-related changes in the function of hemopoietic stroma in mice.

Hemopoietic function of the marrow and splenic stroma in C57BL/6 mice aging from 2 to 24 months were evaluated with the use of subcutaneous implantation method. Although there was no significant difference in the concentration as well as the total number of CFU(S) in femoral marrows between aged and young mice, the rates of CFU(S) repopulation in the femoral marrows from aged donors were significantly lower than in those from young donors when grafted subcutaneously in the 2 month-old syngeneic hosts. On the other hand, the femoral marrow grafts from 2 month-old donors were constantly repopulated irrespective of the age of the hosts. An essentially similar finding was obtained in the study of spleen grafts. It is suggested that the defective hemopoiesis, if any, in aged mice is primarily caused by the changes in hemopoietic stroma rather than those in hemopoietic stem cells.

Maturation sequence hierarchy of murine macrophage progenitor cells.

The heterogeneity of murine macrophage progenitors was analyzed with special interest in their maturation sequence. The gross appearance of macrophage colonies formed by C57BL/6 marrow cells was classified into three types: compact, intermediate, and dispersed. Progenitors of each type could be segregated by sedimentation velocity. Findings of cell-cycle studies showed that different types of colonies were not derived from a single progenitor cell at different times in the cell cycle. Therefore, it is considered that the difference in colony types can be attributed to the heterogeneity of progenitors. Suspension cultures of fractionated marrow cells rich in compact, intermediate, and dispersed colonies yielded mainly intermediate colonies, dispersed colonies, and clusters, respectively. As suspension cultures containing colony stimulating factor were considered to induce cell division as well as maturation, it is supposed that progenitors of compact, intermediate, and dispersed colonies constitute a maturation sequence hierarchy in this order.

Achievement of a complete cytogenetic response with hydroxyurea in a patient with chronic myelogenous leukemia.

Hydroxyurea rarely produces a complete cytogenetic remission in patients with chronic myelogenous leukemia (CML). In this report, we describe one case of the CML patient who achieved complete cytogenetic remission (no Ph chromosome in 20-25 metaphase cells) by treatment with hydroxyurea alone. By the fluorescent in situ hybridization (FISH) methodology using bcr/abl specific translocation probe, sequential bone marrow specimens from the patient showed the characteristic 9;22 translocation at a higher rate (9-10%) than the normal control range (2.49-4.88%) at the time of complete cytogenetic remission. Thus, it is suggested that FISH is a more sensitive method to detect the bcr/abl fusion gene than conventional cytogenetic analysis for the detection of minimal residual disease in CML.

The dual expression of minor and major bcr/abl chimeric mRNA in blast crisis of chronic myelogenous leukemia.

The hypothesis that minor bcr/abl fusion mRNA is produced in blast crisis of chronic myelogenous leukemia (CML) is examined. The RNA transcripts encoding the minor and major bcr/abl fused protein were detected by polymerase chain reaction (PCR) using RNA from peripheral blood or bone marrow cells of eight patients with blast crisis or accelerated phase of CML. The mRNA encoding for major bcr/abl was detected in all eight cases. In four patients, however, transcripts encoding for minor bcr/abl mRNA were detected, as well as major bcr/abl mRNA. The presence of minor bcr/abl mRNA was verified with the hybridization with a junction-specific probe and DNA sequencing analysis of PCR products. The appearance of minor bcr/abl fusion mRNA was associated with the lymphoblastic immunophenotype of the blast cells. In two of these four patients, samples of initial diagnosis of chronic phase of CML were available, which did not show minor bcr/abl transcript. We conclude that the appearance of minor bcr/abl mRNA transcript is associated with the terminal evolution of CML in lymphoblastic crisis.

Differential expression patterns of three glutamate transporters (GLAST, GLT1 and EAAC1) in the rat main olfactory bulb.

Glutamate is the main neurotransmitter in the olfactory bulb. Therefore, glutamate transporters, which regulate the concentration of extracellular glutamate, might play pivotal roles in odor processing. In this study, we examined expressions of three glutamate transporters (GLAST, GLT1 and EAAC1) in the olfactory bulb using in situ hybridization and immunohistochemistry. EAAC1 mRNA was expressed in neurons, such as periglomerular cells, tufted cells, mitral cells and granule cells as shown before in other brain areas. In contrast, GLAST and GLT1 were found in glial cells throughout the olfactory bulb, with intenser expressions in the glomerular layer, external plexiform layer and internal plexiform layer where glutamatergic synapses are concentrated. In addition, using double staining immunohistochemistry we clearly showed that GLAST and GLT1 were expressed in astrocytes. Furthermore, we found that GLAST was also intensely expressed in the subependymal layer where precursor cells exist. These results suggest each glutamate transporter plays its unique role not only in glutamatergic neurotransmission but also in cell differentiation and migration in the olfactory bulb.

Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity.

We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.

Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature.

To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermo-responsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32 degrees C, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37 degrees C, as on the nongrafted culture dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20 degrees C without need for dispase. No cell remnants were observed on the dishes. Such cell sheet detachment was not observed on nongrafted dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted dishes. Transmission electron microscopy revealed that desmosomes were destroyed in dispase treatment but retained in low-temperature treatment. Use of thermo-responsive dishes was examined as a new tool for tissue engineering to achieve the preparation of artificial epithelium for cell transplantation as well as for the investigation of intact multilayered keratinocyte sheets.

Effects of prostaglandin E2 on the ultrastructure of the golden hamster parathyroid gland.

The effects of different ages on large vacuolar bodies in the parathyroid glands of golden hamsters after administration of prostaglandin E2 (PGE2) were investigated. In the parathyroid glands of the young and senile animals 15 min and the senile animals 60 min after administration of PGE2, the mean serum calcium concentration was significantly higher when compared to that of the young and senile control animals, respectively. In the experimental adult animals 60 min after administration of PGE2, the serum calcium concentration was seen to increase. In the parathyroid glands of the young animals 15 min and the adult and senile animals 60 min after administration of PGE2, the percent area occupied by large vacuolar bodies was significantly increased when compared to that of the young, adult and senile control animals, respectively. These findings suggest that the percent area occupied by large vacuolar bodies is increased in response to hypercalcemia induced by PGE2. It is thought that in the parathyroid glands suppressed by hypercalcemia there is a relationship between the percent area occupied by large vacuolar bodies and aging.

Effects of melatonin on the ultrastructure of the golden hamster parathyroid gland.

Ultrastructural changes of the parathyroid glands of melatonin-treated golden hamsters were studied. Many chief cells in the parathyroid glands after 1 hour of administration of melatonin contained poorly-developed Golgi complexes associated with a few prosecretory granules and numerous lipid droplets as compared with those of the control animals. The morphology of the parathyroid glands after 5 hours of administration resembled that of the control animals. Many chief cells in the parathyroid glands after 24 hours of administration had well-developed Golgi complexes and cisternae of the granular endoplasmic reticulum, numerous prosecretory granules, a few lipid droplets and many secretory granules in the peripheral cytoplasm as compared with those of the control animals. The ultrastructure of the parathyroid glands after 48 hours of administration was almost similar to that of the control animals. It is considered that melatonin affects the secretory activity of the parathyroid gland.

Ultrastructure of mass of floccular substance in the parathyroid gland of golden hamster.

Mass of floccular substance was observed in the parathyroid glands of fetal, newborn and infantile golden hamsters. Mass composed of floccular substances was spherical with no limiting membrane around it. It was located near the nucleus and the Golgi area, but was also observed in the peripheral cytoplasm. No cell organelles were detected within area of mass.

Ultrastructural study on the effects of hypophysectomy on the golden hamster parathyroid gland.

The ultrastructure of the parathyroid glands of hypophysectomized golden hamsters was studied. In the parathyroid glands of hypophysectomized animals the Golgi complexes and secretory granules were significantly decreased and large vacuolar bodies were significantly increased compared with those of the control animals. In addition, the chief cells contained a few prosecretory granules in the Golgi areas and a few secretory granules were present in the peripheral cytoplasm. These results suggest that the synthesis and release of parathyroid hormone may be suppressed in the parathyroid glands of the hypophysectomized animals.

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland.

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.