Efficiency of diversion of the first aliquot of blood and prestorage leukoreduction for preventing bacterial contamination in red blood cell concentrates assessed using a rapid polymerase chain reaction-based bacterial detection system.

OBJECTIVES: Sepsis caused by the bacterial contamination of blood products is a major infection risk associated with blood transfusion. Diversion of the initial 25 mL of blood and prestorage leukoreduction were implemented in Japan in 2007 for all donated blood products. We assessed the efficacy of these new collection procedures in preventing bacterial contamination of red blood cell (RBC) concentrates. METHODS: Broad-range 16S ribosomal RNA polymerase chain reaction was used to determine bacterial contamination in segment samples of RBCs before and after implementation of the new collection procedures. To evaluate whether these new procedures reduced bacterial contamination, we compared bacterial contamination rates of blood samples from diversion pouches with those of segment samples from the same donor's RBCs. RESULTS: The rate of bacterial contamination of RBCs before implementation of the new collection procedures was 1.27%. Most of the isolated bacteria were Staphylococcus epidermidis or Propionibacterium acnes. After implementation, this rate was significantly reduced to 0.10%. Of the 233 whole blood samples obtained from the Mie Red Cross Blood Center, 1.72% of blood samples from diversion pouches were contaminated, but no bacterial contamination was detected in segment samples from the same donor's RBCs after prestorage leukoreduction. CONCLUSIONS: The new collection procedure significantly reduced bacterial contamination of RBC concentrates.

Topological transitions among skyrmion- and hedgehog-lattice states in cubic chiral magnets.

Manipulating topological spin textures is a key for exploring unprecedented emergent electromagnetic phenomena. Whereas switching control of magnetic skyrmions, e.g., the transitions between a skyrmion-lattice phase and conventional magnetic orders, is intensively studied towards development of future memory device concepts, transitions among spin textures with different topological orders remain largely unexplored. Here we develop a series of chiral magnets MnSi1-xGex, serving as a platform for transitions among skyrmion- and hedgehog-lattice states. By neutron scattering, Lorentz transmission electron microscopy and high-field transport measurements, we observe three different topological spin textures with variation of the lattice constant controlled by Si/Ge substitution: two-dimensional skyrmion lattice in x = 0-0.25 and two distinct three-dimensional hedgehog lattices in x = 0.3-0.6 and x = 0.7-1. The emergence of various topological spin states in the chemical-pressure-controlled materials suggests a new route for direct manipulation of the spin-texture topology by facile mechanical methods.

Monitoring of hemostatic abnormalities in major orthopedic surgery patients treated with edoxaban by APTT waveform.

INTRODUCTION: An analysis of the activated partial thromboplastin time (APTT) in major orthopedic surgery patients receiving edoxaban for the prevention of venous thromboembolism (VTE) was carried out. METHODS: The APTT waveform was analyzed in the above patients to monitor edoxaban administration. RESULTS: Of these 99 patients, 12 exhibited deep vein thrombosis, and 25 had massive bleeding. An increased biphasic pattern of the APTT waveform was observed after the administration of edoxaban, but there were no significant differences between the patients with and without complications. The peak times of acceleration, velocity, and 1/2 fibrin formation were significantly prolonged after the administration of edoxaban, especially in patients with massive bleeding, and were moderately correlated with the anti-Xa activity. While the heights of velocity and acceleration peak 2 were lower in patients receiving warfarin treatment than in those receiving edoxaban, the widths of these parameters were significantly longer. The height of 1/2 fibrin formation and the width of acceleration peaks 1 and 2 and the velocity were significantly increased after the administration of edoxaban. CONCLUSION: The peak time of the APTT waveform was significantly prolonged after the administration of edoxaban. The analysis of the APTT waveform may therefore be useful for the prediction of the risk of massive bleeding.

Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus is associated with impaired afferent arteriolar responsiveness to opening of voltage-gated calcium channels. Diabetes was induced by injection of streptozocin (65 mg/kg, i.v.) and insulin was administered via an osmotic minipump to achieve moderate hyperglycemia. Sham rats received vehicle treatments. 2 wk later, the in vitro blood-perfused juxtamedullary nephron technique was used to allow videomicroscopic measurement of afferent arteriolar contractile responses to increasing bath concentrations of either Bay K 8644 or K+. Baseline afferent arteriolar diameter in kidneys from diabetic rats (26.4+/-1.2 microm) exceeded that of Sham rats (19.7+/-1.0 microm). Bay K 8644 evoked concentration-dependent reductions in afferent diameter in both groups of kidneys; however, arterioles from Sham rats responded to 1 nM Bay K 8644 while 100 nM Bay K 8644 was required to contract arterioles from diabetic rats. The EC50 for K+-induced reductions in afferent arteriolar diameter was greater in diabetic kidneys (40+/-4 mM) than in kidneys from Sham rats (28+/-4 mM; P < 0.05). In afferent arterioles isolated by microdissection from Sham rats and loaded with fura 2, increasing bath [K+] from 5 to 40 mM evoked a 98+/-12 nM increase in intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i responses to 40 mM K+ were suppressed in afferent arterioles from diabetic rats (delta = 63+/-5 nM), but were normalized by decreasing bath glucose concentration from 20 to 5 mM. These observations indicate that the early stage of insulin-dependent diabetes mellitus is associated with a functional defect in afferent arteriolar L-type calcium channels, an effect which may contribute to suppressed afferent arteriolar vasoconstrictor responsiveness and promote glomerular hyperfiltration.

Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins.

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.

Peptide-based bovine leukemia virus (BLV) vaccine that induces BLV-Env specific Th-1 type immunity.

In controlling retrovirus infection and replication, cell-mediated immunity (CMI) is considered to be effective. To develop a synthetic peptide vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed and inoculated to BALB/c mice. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses, and a weak cytotoxic lymphocyte response. The spleen cells from the immunized mice produced a large amount of IFN-gamma and IL-2, whereas they released neither IL-4 or IL-10. Mannan-coated liposome containing two kinds of peptides (the 121-140 and 142-161 regions of BLV Env gp51) also induced peptide-specific lymphocyte proliferative response and IFN-g production in C57BL/6 mice. Thus, the synthetic T-cell epitope peptide-liposome system augmented a strong Th-1 type immunity in mice.

Stage-specific cell-cycling of hematopoietic progenitor cells.

We studied the growth of hematopoietic progenitors at different progressive stages of differentiation and focused especially on changes in cell-cycling. Hematopoietic progenitors from 5-fluorouracil (5-FU)-treated mice were separated into three groups on the basis of differentiation, Stages I, II, and III, and have studied their cell-cycling. Primary marrow cells collected from 5-FU-treated mice were categorized as Stage I progenitors. Stages II and III progenitors are early and late progenies of Stage I progenitors, respectively. The rate of growth of hematopoietic progenitors supported by interleukin-3 (IL-3) and steel factor (SF) was estimated by sequential analysis of colony formation and studying replating efficiency of individual colonies. The time required for hematopoietic progenitors to go through the cell-cycle shortened as their stage of differentiation progressed. Similar results were obtained with other growth factor combinations. The analysis of DNA content of cells suggests that shortening of cell-cycling is mainly due to a decrease in the time of G1 phase of the cell-cycle. Our results demonstrate that in early hematopoiesis, the cell-cycling of hematopoietic progenitors accelerates as they differentiate.

Frequent gene expression of granulocyte colony-stimulating factor (G-CSF) receptor in CD7+ surface CD3- acute lymphoblastic leukaemia.

Gene expression of various cytokine receptors in CD7+ acute lymphoblastic leukemia (ALL) cells in relation to responsiveness to these cytokines was examined by reverse transcription polymerase chain reaction and Northern blot studies. Leukemic cells from all of seven CD7+ ALL patients examined fulfilled the criteria for ALL according to the FAB classification; surface CD3 was absent in all of these patients, while cytoplasmic CD3 and/or CD3 epsilon mRNA were found in all of them. Samples from six of the seven patients at initial disease expressed the granulocyte colony-stimulating factor receptor (G-CSFR) gene. Leukemic cells with G-CSFR transcripts from one patient at initial disease showed growth response to G-CSF in vitro, and those from two other patients became responsive to G-CSF at relapse. Neither in vitro nor in vivo myeloid differentiation was observed in any samples that responded to G-CSF. Interleukin 3R alpha (IL-3R alpha) gene was expressed in samples from one patient at initial disease and from two patients at relapse. GM-CSFR beta gene mRNA was detected in two patients with IL-3R alpha mRNA. Our results show that the leukemic cells in these CD7+ ALL patients frequently expressed G-CSFR as a functional property, thus calling attention to the appropriate clinical application of G-CSF for ALL patients.

Staggered magnetism in LiV(2)O(4) at low temperatures probed by means of the muon Knight shift.

We report on measurement of the muon Knight shift in single crystals of LiV(2)O(4). Contrary to what is anticipated for the heavy fermion state based on the Kondo mechanism, the presence of inhomogeneous local magnetic moments is demonstrated by the broad distribution of the Knight shift at temperatures well below the presumed 'Kondo temperature' ([Formula: see text] K). Moreover, a significant fraction ([Formula: see text]) of the specimen gives rise to a second component which is virtually non-magnetic. These observations strongly suggest that the anomalous properties of LiV(2)O(4) originate from frustration of local magnetic moments.

Uniaxial stress control of skyrmion phase.

Magnetic skyrmions, swirling nanometric spin textures, have been attracting increasing attention by virtue of their potential applications for future memory technology and their emergent electromagnetism. Despite a variety of theoretical proposals oriented towards skyrmion-based electronics (that is, skyrmionics), few experiments have succeeded in creating, deleting and transferring skyrmions, and the manipulation methodologies have thus far remained limited to electric, magnetic and thermal stimuli. Here, we demonstrate a new approach for skyrmion phase control based on a mechanical stress. By continuously scanning uniaxial stress at low temperatures, we can create and annihilate a skyrmion crystal in a prototypical chiral magnet MnSi. The critical stress is merely several tens of MPa, which is easily accessible using the tip of a conventional cantilever. The present results offer a new guideline even for single skyrmion control that requires neither electric nor magnetic biases and consumes extremely little energy.

Increased level of salivary prostaglandins in patients with major depression.

We quantified the amounts of salivary prostaglandin (PG) D2, PGE2, and PGF2 alpha by radioimmunoassay in 32 patients with major depressive disorder, 16 patients with minor depressive disorder, 24 patients with neurotic disorders (panic, generalized anxiety, phobic, somatization, and obsessive compulsive), and 28 healthy controls. In the saliva of patients with major depressive disorder, the concentrations of immunoreactive PGs (PGD2, 385 +/- 71 pg/ml; PGE2, 498 +/- 105 pg/ml; PGF2 alpha, 444 +/- 100 pg/ml) were significantly higher than those of the healthy controls (PGD2, 129 +/- 18 pg/ml; PGE2, 207 +/- 25 pg/ml; PGF2 alpha, 164 +/- 17 pg/ml). On the other hand, the salivary concentrations of immunoreactive PGs from patients with minor depressive disorder or neurotic disorders were comparable to those of the controls. These results suggest that the level of salivary PGs may be an indicator of major depressive disorder.

Salivary prostaglandin concentrations: possible state indicators for major depression.

Salivary prostaglandin concentrations were determined in 42 patients with major depressive disorder, 16 patients with minor depressive disorder, and 39 healthy control subjects. The diagnoses were made according to the Research Diagnostic Criteria. The patients with major depressive disorder had higher salivary prostaglandin concentrations than the control subjects, but the patients with minor depressive disorder did not. Furthermore, the salivary prostaglandin concentrations of the patients with major depressive disorder showed a high correlation with the severity of the depression. These results suggest that high salivary prostaglandin concentrations may be state indicators for major depression.

Oligomannose-coated liposomes as an adjuvant for the induction of cell-mediated immunity.

The effect of the coating of ovalbumin-reconstituted liposomes with various oligosaccharides on their immunogenicity was investigated in mice. The coating of liposomes with oligomannose or yeast mannan drastically enhanced their ability to induce an ovalbumin-specific delayed-type footpad swelling response with a peak at 24 to 48 h post-challenge. Among various oligosaccharides tested, only those with mannose residue at the nonreducing termini manifested the activity when applied to liposomes. Since such oligosaccharides are ubiquitously found in the body, these results suggested the usefulness of oligomannose-coated liposomes as a safe adjuvant for the induction of cell-mediated immunity.

Exaggerated messenger RNA expression of inflammatory cytokines in human T-cell lymphotropic virus type I-associated myelopathy.

OBJECTIVE: To investigate the expression of inflammatory cytokine messenger RNA (mRNA) in peripheral blood mononuclear cells of patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM). PATIENTS: Seventeen patients with HAM, 18 HTLV-I-seropositive carriers, and 10 seronegative individuals were studied. MAIN OUTCOME MEASURE: We compared the expression of tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha (IFN-alpha), IFN-beta, and IFN-gamma, and interleukin 1 alpha (IL-1 alpha) and IL-1 beta by reverse transcriptase-polymerase chain reaction. RESULTS: In patients with HAM, the reverse transcriptase-polymerase chain reaction products of TNF-alpha, GM-CSF, IFN-gamma, and IL-1 alpha were detected in significantly higher incidences than in HTLV-I-seropositive carriers and seronegative controls. Furthermore, simultaneous mRNA expression of three or more of these four cytokines was detected in all patients with HAM compared with only 21.4% of HTLV-I-seropositive carriers. By contrast, there was no significant difference in mRNA expression of IFN-alpha, IFN-beta, and IL-1 beta among patients with HAM, HTLV-I-seropositive carriers, and HTLV-I-seronegative controls. CONCLUSIONS: An exaggerated mRNA expression of several inflammatory cytokines, including TNF-alpha, GM-CSF, IFN-gamma, and IL-1 alpha, was demonstrated in peripheral blood mononuclear cells of patients with HAM. Moreover, transcripts of these cytokines were simultaneously up-regulated in patients with HAM, suggesting that an inflammatory state in the central nervous system may be related to the pathogenesis of HAM.

H-31 human breast cancer cells stimulate type I collagenase production in osteoblast-like cells and induce bone resorption.

Bone is one of the most common sites of metastasis in breast cancer. For metastasis to occur in bone, tumor cells must induce osteolysis by osteoclasts. Degradation of the osteoid layer by type I collagenase is a necessary process before osteolysis can occur because the osteoid layer hinders osteoclasts from adhering to bone. In this study, we investigated the function of H-31 human breast cancer cells in inducing type I collagenase production and in enhancing bone resorption. H-31 cells did not themselves produce type I collagenase whereas MG-63 human osteoblast-like cells and MC3T3-E1 mouse osteoblast cells constantly produced type I collagenase. When these osteoblast-like cells were cocultured with H-31 cells, type I collagenase production was enhanced. The same enhancement occurred when the conditioned medium of H-31 cells was added to the osteoblast-like cells. The activity of this type I collagenase was inhibited by EDTA and minocyclin, an inhibitor of matrix metalloproteinases, hence it was identified as matrix metalloproteinase-1 (MMP-1). H-31 cells exhibited chemotactic migration towards collagen; therefore, collagen degraded by MMP-1 may play an important role in the localisation of breast cancer cells like H-31 to bone. In an organ culture system using newborn mouse calvaria, the conditioned medium of H-31 cells increased the concentration of calcium in the medium, and this effect was inhibited by minocyclin, indicating that bone resorption occurred in this system. Based on these observations, we speculate that type I collagenase produced by osteoblast cells in response to breast cancer cells (exemplified by H-31) may facilitate degradation of the osteoid layer and the homing of breast cancer cells to bone. This can lead to osteolysis by osteoclasts, a crucial event for bone metastasis.

Stimulatory effects of phenytoin on osteoblastic differentiation of fetal rat calvaria cells in culture.

Phenytoin (diphenylhydantoin, DPH), an anticonvulsant drug for epileptic patients, has several adverse effects, including calvarial thickening and coarsening of the facial features, which occur with chronic DPH therapy. While previous studies have demonstrated that DPH has an anabolic action on bone cells in vivo and in vitro, the basis of these effects is not fully understood. In this study, the effect of DPH on osteoblastic differentiation of fetal rat calvaria (RC) cells in culture was investigated by measuring bone nodule (BN) formation, cell growth, alkaline phosphatase (ALPase) activity, collagen synthesis, and expression of osteocalcin (OC) and osteopontin (OP) mRNAs. Continuous treatment of RC cells with DPH for 18 days dose-dependently increased the mineralized BN number by 1.2-1.7-fold at concentrations of 12.5-200 micromol/L DPH. Cell growth was not affected at the same concentrations of DPH. ALPase activity was stimulated by DPH (1.1-1.9-fold) dose-dependently and was maintained at higher levels in DPH-treated cells throughout the experimental period. DPH increased mineralized and unmineralized BN formations both in the presence and the absence of 10(-8) mol/L dexamethasone (Dex). Expression of OC and OP mRNAs was markedly augmented by DPH on days 12-24 and on days 12-18, respectively. While control mRNA levels of OC and OP increased with time, the increases in DPH-treated cells were greater than those of the controls and the stimulatory effects were dose-dependent. Type I collagen was also influenced by DPH; mRNA level was enhanced and the percentage of collagen synthesized was increased significantly, by 200 micromol/L DPH. When DPH was added in three different culture stages, days 1-6 (growth), days 7-12 (matrix development), and days 13-18 (mineralization), BN formation was influenced primarily on days 1-6 and secondarily on days 7-12, but not on days 13-18, suggesting that DPH increased BN formation by enhancing not only the proportion of osteoprogenitor cells in the early stage but also the proportion of functional osteoblasts in the middle stage within mixed-cell populations. Moreover, such increases were detected in conditions of both Dex(+) and Dex(-). These findings demonstrate that DPH stimulates osteoblast-associated markers such as BNs, ALPase, OC, OP, and type I collagen by continuously affecting the stages of growth and matrix development in RC cells, and suggests that the stimulatory effects by DPH may possibly be induced independent of those by Dex.

Novel combretastatin analogues effective against murine solid tumors: design and structure-activity relationships.

A series of combretastatin A-4 (CA-4) analogues were synthesized, and their cytotoxic effects against murine Colon 26 adenocarcinoma and inhibitory activity on tubulin polymerization were evaluated. Since CA-4 has limited aqueous solubility, the target compounds were designed to improve solubility by introduction of a nitrogen-containing group. Among the compounds synthesized, those with an amino moiety in place of the phenolic OH of CA-4 showed potent antitubulin activity and cytotoxicity against murine Colon 26 adenocarcinoma in vitro. Some of the compounds which were potent in vitro were evaluated in the murine tumor model Colon 26 in vivo. Among these, 13bHCl, 21aHCl, and 21bHCl showed significant antitumor activity in the animal model, while CA-4 was ineffective. 13bHCl and 21aHCl were further evaluated in two murine tumor models (Colon 38 and 3LL) and human xenografts HCT-15. These compounds showed potent antitumor activity comparable or superior to that of CDDP. The structure-activity relationships of this series of compounds are also discussed.