Differential interference contrast microscopy for cells using hard x-ray holography.

We propose a differential interference contrast method for cells using hard x-ray Gabor holography and knife-edge filtering in the spatial frequency domain, without relying on beam shearing. A phase object is holographically recorded and reconstructed by computer. Interference between the wavefronts of zeroth order weighted by ejπ/2 in the positive frequency region produces a dark image. Similarly, interference between the wavefronts of the zeroth order weighted by ej3π/2 in the negative frequency region produces a bright image. By adding these two intensity distributions, good quality phase-contrast images of 8-µm-diameter polystyrene beads and human HeLa cells were obtained.

Efficacy of stem cells on periodontal regeneration: Systematic review of pre-clinical studies.

This systematic review aims to evaluate mesenchymal stem cells (MSC) periodontal regenerative potential in animal models. MEDLINE, EMBASE and LILACS databases were searched for quantitative pre-clinical controlled animal model studies that evaluated the effect of local administration of MSC on periodontal regeneration. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines. Twenty-two studies met the inclusion criteria. Periodontal defects were surgically created in all studies. In seven studies, periodontal inflammation was experimentally induced following surgical defect creation. Differences in defect morphology were identified among the studies. Autogenous, alogenous and xenogenous MSC were used to promote periodontal regeneration. These included bone marrow-derived MSC, periodontal ligament (PDL)-derived MSC, dental pulp-derived MSC, gingival margin-derived MSC, foreskin-derived induced pluripotent stem cells, adipose tissue-derived MSC, cementum-derived MSC, periapical follicular MSC and alveolar periosteal cells. Meta-analysis was not possible due to heterogeneities in study designs. In most of the studies, local MSC implantation was not associated with adverse effects. The use of bone marrow-derived MSC for periodontal regeneration yielded conflicting results. In contrast, PDL-MSC consistently promoted increased PDL and cementum regeneration. Finally, the adjunct use of MSC improved the regenerative outcomes of periodontal defects treated with membranes or bone substitutes. Despite the quality level of the existing evidence, the current data indicate that the use of MSC may provide beneficial effects on periodontal regeneration. The various degrees of success of MSC in periodontal regeneration are likely to be related to the use of heterogeneous cells. Thus, future studies need to identify phenotypic profiles of highly regenerative MSC populations.

Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

I-ROAD could be efficient in predicting severity of community-acquired pneumonia or healthcare-associated pneumonia.

INTRODUCTION: The ability to predict the prognosis of patients with pneumonia is critical, especially when making decisions regarding treatment regimens and sites of care. However, prognostic guidelines for healthcare-associated pneumonia (HCAP) have yet to be established. I-ROAD is the prognostic guideline of the Japanese Respiratory Society for hospital-acquired pneumonia (HAP). This study compared available prognostic guidelines to determine the usefulness of I-ROAD as a prognostic tool for patients with HCAP. METHODS: We conducted a retrospective review of all patients with pneumonia admitted to Kameda Medical Center, Japan, from January 2006 to September 2009. Patients were categorised into two groups, namely those with community acquired pneumonia (CAP) and those with HCAP. We compared the baseline characteristics, laboratory findings, identified pathogens, antibiotic regimens, clinical outcomes, pneumonic severity and prognostic accuracy of each guideline between the two patient groups. The severity of each disease was assessed on admission using the A-DROP, CURB-65, PSI and I-ROAD guidelines. RESULTS: Of the 302 patients evaluated, 228 (75.5%) were diagnosed with CAP and 74 (24.5%) with HCAP. Patients with HCAP were older and had a higher performance status than patients with CAP. The mortality rate in the CAP group tended to rise with increasing severity scores of prognostic guidelines. Although the severity scores of all prognostic guidelines could predict 30-day mortality in patients with CAP, I-ROAD exhibited a higher discriminatory power for patients with HCAP based on analysis of receiver-operating characteristic curves. CONCLUSION: I-ROAD could be more accurate than other prognostic guidelines for evaluating the severity of HCAP.

Severe congenital factor XIII deficiency caused by novel W187X and G273V mutations in the F13A gene; diagnosis and classification according to the ISTH/SSC guidelines.

Factor XIII (FXIII) consists of the A and B subunits (FXIII-A and FXIII-B) and stabilizes fibrin clots. Defects in either the FXIII-A or FXIII-B gene lead to congenital FXIII deficiency, which manifests a life-long haemorrhagic tendency. Thus, prophylactic FXIII replacement therapy is recommended. To establish a management plan for a 30-year-old male patient with 'indefinite' FXIII deficiency (<40% of the normal FXIII), he was characterized by state-of-the-art techniques as guided by the FXIII/Fibrinogen subcommittee of ISTH/SSC. FXIII activity turned out to be virtually undetectable by three functional assays. Four immunological assays detected essentially no FXIII protein, FXIII-A antigen, and A2 B2 antigen, but normal FXIII-B antigen. Accordingly, he was diagnosed as a 'severe' FXIII-A deficiency case. He had no anti-FXIII antibodies, because a 1:1 cross-mixing test (ammonia release assay) and a five-step mixing test (amine incorporation assay) between his plasma and normal plasma demonstrated deficiency patterns. Furthermore, a dosing test using plasma-derived FXIII concentrates revealed its normal recovery. DNA sequencing analysis identified two novel mutations, W187X & G273V, in the F13A gene. Genetic analyses confirmed that he was a compound heterozygote and his mother and sister were heterozygotes of either one of these mutations, indicating the hereditary nature of this disorder. Molecular modelling predicted that the G273V mutation would cause clashes with the surrounding residues in the core domain of FXIII-A, and ultimately would result in the instability of the mutant molecule. Detailed characterization of 'indefinite' FXIII deficiency made it possible to make its definite diagnosis and best management plan.

Direct measurement of speed of sound in cartilage in situ using ultrasound and magnetic resonance images.

This study verified the accuracy of the speed of sound (SOS) measured by the combination method, which calculates the ratio between the thickness values of cartilage measured by using the magnetic resonance imaging (MRI) and the ultrasonic pulse-echo imaging, and investigated in vivo application of this method. SOS specific to an ultrasound imaging device was used as a reference value to calculate the actual SOS from the ratio of cartilage thicknesses obtained from MR and ultrasound images. The accuracy of the thickness measurement was verified by comparing results obtained using MRI and a non-contact laser, and the accuracy of the calculated SOS was confirmed by comparing results of the pulse-echo and transmission methods in vitro. The difference between laser and MRI measurements was 0.05 ± 0.22 mm. SOS values in a human knee measured by the combination method in the medial and lateral femoral condyles were 1650 ± 79 and 1642 ± 78 m/s, respectively (p < 0.05). The results revealed the feasibility of in situ SOS measurement using the combination method.

Comprehensive diagnostic ability of endocytoscopy compared with biopsy for colorectal neoplasms: a prospective randomized noninferiority trial.

BACKGROUND AND STUDY AIMS: Endocytoscopy enables observation at 450-fold magnification during gastrointestinal endoscopy, allowing on-site "optical biopsy." We compared the accuracies of endocytoscopy and standard biopsy for the diagnosis of colorectal neoplasms. PATIENTS AND METHODS: We performed a randomized, controlled, open-label trial of patients with colorectal lesions (≥ 5 mm) detected during colonoscopy in a tertiary referral center. We randomly assigned the 203 detected lesions of 170 eligible patients to either the endocytoscopy or standard biopsy group. An on-site endoscopist assessed the histopathology of the endocytoscopy group lesions according to the endocytoscopic findings, whereas a pathologist later assessed standard biopsy group lesions by microscopic examination of the biopsy specimens. We calculated the diagnostic accuracies in both groups with reference to the final histopathology of the resected specimens. The primary endpoint was to determine whether the diagnostic accuracy of endocytoscopy for neoplastic lesions was noninferior to that of standard biopsy (with a predefined noninferiority margin of 10%). Analyses were by intention-to-treat and per-protocol. The study is registered, number UMIN000003923. RESULTS: Overall, 102 lesions in the endocytoscopy group and 101 in the standard biopsy group were available for primary outcome analysis. There were no complications. The diagnostic accuracy of endocytoscopy for the discrimination of neoplastic lesions was 94.1% (95% confidence interval 87.6% to 97.8%), whereas that of standard biopsy was 96.0% (90.2% to 98.9%), which is within the noninferiority margin (absolute difference -1.9%, -8.6% to +5.0%). CONCLUSIONS: Endocytoscopy is noninferior to standard biopsy for the discrimination of neoplastic lesions. With its advantage of providing an on-site diagnosis, endocytoscopy could provide a novel alternative to standard biopsy in routine colonoscopy.

Antitumor efficacy of oncolytic herpes simplex virus adsorbed onto antigen-specific lymphocytes.

Although several studies have reported that locally administering oncolytic viruses effectively targets malignancies, the efficacy of systemically administered oncolytic viruses is restricted. Recently, however, it was reported that systemic administration of oncolytic vesicular stomatitis virus adsorbed onto antigen-specific lymphocytes was effective against malignancies. We hypothesized that intravenously administering such virus might have significant potential in treatment of the malignant tumors. We adsorbed oncolytic herpes simplex virus-1 mutant R3616 onto lymphocytes harvested from mice with acquired antitumor immunity. We administered adsorbed R3616 to peritoneally disseminated tumors and analyzed the efficacy of this treatment. Mice administered adsorbed R3616 survived significantly longer than mice administered R3616 adsorbed onto non-specific lymphocytes, or mice administered either virus or tumor antigen-specific lymphocytes alone. In this context, herpes oncolytic virus is a promising treatment not only for primary lesions, but also for multiple metastasizing lesions. This treatment strategy may become one of the most effective methods for systemic virus delivery.

Oxidative stress levels in myelodysplastic syndrome patients: their relationship to serum ferritin and haemoglobin values.

Reactive oxygen species (ROS) and serum ferritin levels are both considered to be important biological factors in the pathogenesis of myelodysplastic syndrome (MDS). This study evaluated the levels of ROS in 40 patients with MDS (19 males and 21 females) using the Free Radical Analytical System, FRAS4, and derivatives of reactive oxygen metabolite kits. The patients' mean age was 67.3 years (range 58 - 86 years). The sera of 34 (85%) patients exhibited higher levels of oxidative stress than the reference range. There was a positive correlation between ROS levels and serum ferritin levels, and a negative correlation between ROS levels and haemoglobin levels. There was a negative relationship between serum haemoglobin and ferritin levels. The results indicated that iron accumulation or severe anaemia could contribute to oxidative stress in MDS patients. Iron chelation and antioxidant therapy may be suitable for the management of MDS.

A phase I dose-escalation clinical trial of intraoperative direct intratumoral injection of HF10 oncolytic virus in non-resectable patients with advanced pancreatic cancer.

In 2005, we initiated a clinical trial that examined the efficacy of the oncolytic virus HF10 to treat pancreatic cancer. Pancreatic cancer continues to have a high mortality rate, despite multimodal treatments for patients, and new therapeutic methods are greatly needed. The current mainstream methods for cancer treatment include biological therapeutics such as trastuzumab (Herceptin) for breast cancer or erlotinib (Tarceva) for non-small cell lung cancer. Oncolytic virus therapy is a new and promising treatment strategy for cancer. Oncolytic viruses are novel biological therapeutics for advanced cancer that appear to have a wide spectrum of anticancer activity with minimal human toxicity. To examine the efficacy of oncolytic virus therapy for pancreatic cancer, we initiated pilot studies by injecting six patients with non-resectable pancreatic cancer with three doses of HF10. All patients were monitored for 30 days for local and systemic adverse effects and were not administered any other therapeutics during this period. There were no adverse side-effects, and we observed some therapeutic potential based on tumor marker levels, survival, pathological findings and diagnostic radiography. The tumors were classified as stable disease in three patients, partial response in one patient and progressive disease in two patients.

Phorbol 12,13-dibutyrate-mediated contraction of the stimulated bronchial smooth muscles of mice.

Increasing evidence suggests that protein kinase C (PKC) is involved in the Ca(2+) sensitization of various smooth muscle contractions. However, the exact role of PKC on bronchial smooth muscle (BSM) contraction is still unclear. In the present study, to determine the role of PKC activation in the BSM contraction, the effects of phorbol 12,13-dibutyrate (PDBu) on BSM tone were examined in the absence and presence of contractile stimulation. Although PDBu had no effect on the basal tone, the contraction induced by acetylcholine, high K(+) depolarization or Ca(2+) ionophore A23187 was significantly augmented by PDBu. Western blot analyses also revealed that the increase in the level of phosphorylated myosin light chain (MLC) induced by high K(+) depolarization was significantly augmented by PDBu treatment. Interestingly, neither high K(+) depolarization alone nor PDBu alone caused CPI-17 phosphorylation, but a significant phosphorylation of CPI-17 was observed when BSMs were co-stimulated by high K(+) and PDBu. Thus, a certain level of intracellular Ca(2+) might be needed both for an activation of CPI-17 and an induction of contraction induced by PDBu in mouse BSMs.

Lovastatin inhibits antigen-induced airway eosinophilia without affecting the production of inflammatory mediators in mice.

OBJECTIVE AND DESIGN: Statins have been proposed as a novel treatment of respiratory diseases. To determine the beneficial effects of statins on allergic bronchial asthma, the effect of systemic treatment with lovastatin on antigen-induced airway inflammation was investigated. SUBJECTS: Male BALB/c mice were used. TREATMENTS: Mice were sensitized and repeatedly challenged with ovalbumin (OA) antigen to induce asthmatic response. Animals were also treated with lovastatin (4 mg/kg/day, i.p.) once a day prior to and during the antigen inhalation period. METHODS: Inflammatory cell counts and levels of interleukin (IL)-4, IL-13, eotaxin, thymus and activation-regulated chemokine and leukotriene B(4) (LTB(4)) in bronchoalveolar lavage (BAL) fluids were measured. RESULTS: Significant increases in eosinophils and levels of the T helper 2 cytokines, chemokines and LTB(4) in BAL fluids in association with the increments of total and OA-specific immunoglobulin E (IgE) in sera were observed in the repeatedly antigen-challenged mice. The airway eosinophilia was ameliorated by lovastatin, whereas it had no significant effect on the levels of these inflammatory mediators or IgE. CONCLUSION: Lovastatin may be beneficial for the treatment of allergic inflammatory diseases in the airways, such as allergic bronchial asthma.

GGTI-2133, an inhibitor of geranylgeranyltransferase, inhibits infiltration of inflammatory cells into airways in mouse experimental asthma.

Statins have been proposed as a novel treatment of respiratory diseases including asthma. Although the mechanism of anti-inflammatory effect of statins is still unclear, an inhibition of protein prenylation by depleting the downstream metabolites of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase might be involved. To test the hypothesis, the effects of GGTI-2133, a direct inhibitor of geran ylgeranyltransferase (GGTase), on antigen-induced airway inflammation were investigated in a murine model of allergic bronchial asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen (OA). Animals were also treated with GGTI-2133 (5 mg/kg/day, i.p.) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused an infiltration of inflammatory cells, especially eosinophils, into airways. Significant increases in interleukin (IL)-4, IL-13, eotaxin, thymus and activation-regulated chemokine (TARC) and leukotriene B4 (LTB4) in bronchoalveolar lavage fluids and total and OA-specific IgE in sera were also found in the antigen-exposed animals. The systemic treatments with GGTI-2133 inhibited the antigen-induced eosinophil infiltration into airways almost completely. However, interestingly, the GGTI-2133 treatment did not affect the levels of these chemotactic factors and IgE. These findings suggest that selective inhibition of GGTase is effective for eosinophilic airway inflammation such as asthma.

Hydration of alcohol clusters in 1-propanol-water mixture studied by quasielastic neutron scattering and an interpretation of anomalous excess partial molar volume.

Quasielastic neutron scattering measurements have been made for 1-propanol-water mixtures in a range of alcohol concentration from 0.0 to 0.167 in mole fraction at 25 degrees C. Fraction alpha of water molecules hydrated to fractal surface of alcohol clusters in 1-propanol-water mixture was obtained as a function of alcohol concentration. Average hydration number N(ws) of 1-propanol molecule is derived from the value of alpha as a function of alcohol concentration. By extrapolating N(ws) to infinite dilution, we obtain values of 12-13 as hydration number of isolated 1-propanol molecule. A simple interpretation of structural origin of anomalous excess partial molar volume of water is proposed and as a result a simple equation for the excess partial molar volume is deduced in terms of alpha. Calculated values of the excess partial molar volumes of water and 1-propanol and the excess molar volume of the mixture are in good agreement with experimental values.

Mass-fractal clustering and power-law decay of cluster size in 1-propanol aqueous solution.

Mesoscale structure of 1-propanol aqueous solutions with propanol mole fraction xp ranging from 0.1 to 0.33 has been studied by means of small angle neutron scattering (SANS) and large-scale reverse Monte Carlo (RMC) technique. Analysis of the SANS intensities in terms of a fractal model shows that the fractal dimension df of mesoscale structure of the solution is about 1.8-1.9 for water-rich solution and about 1.5 for propanol-rich solution. Percolation analysis on the RMC results reveals that the water molecules and the propanol molecules cluster, respectively, as a mass fractal, the dimension dM of which is about 2.3-2.5 for both clusters for water-rich solution. Furthermore, the distribution of the cluster size is expressed by a simple power law with an exponent tau of about 1.35-1.5 for the propanol clusters and 1.05-1.2 for the water clusters. These results imply that the current solution is characterized by polydisperse mass fractals. In fact, a theoretical relation for polydisperse system of mass fractals, df = dM(2-tau), holds well in the current solution. The characteristic change in df from 1.8-1.9 to 1.5 described above is attributed to the crossover between the water-rich regime and the propanol-rich regime. Most of the water molecules and the propanol molecules are located on the interface between clusters, and the water molecules form thin layers of about 10 A thick irrespective of 1-propanol content studied.

GABAA receptor-mediated inhibition by ethanol of long-term potentiation in the basolateral amygdala-dentate gyrus pathway in vivo.

Although ethanol has been reported to inhibit the induction of long-term potentiation in hippocampal CA1 and dentate gyrus synapses of rats, very little is known about the effect of ethanol on synaptic plasticity in other brain regions. Therefore, in the present study, we investigated the effect of ethanol on long-term potentiation in synaptic pathway from the basolateral amygdala to the dentate gyrus by using anesthetized rats in vivo. I.v. (20-40% x 2 ml/kg) or i.c.v. (30-40% x 5 microl) administration of ethanol did not affect the basal amplitude of dentate gyrus field potential evoked by basolateral amygdala stimulation, but significantly inhibited the induction of long-term potentiation following application of tetanic stimulation. Since long-term potentiation in this pathway was independent of N-methyl-d-aspartate receptors, the inhibitory effect of ethanol is unlikely to be caused by suppression of N-methyl-d-aspartate receptor function. Alternatively, long-term potentiation in this pathway was significantly suppressed by the benzodiazepine agonist diazepam (2 mg/kg, i.p.), and the inhibitory effect of ethanol was abolished by the GABAA receptor channel blocker picrotoxin (1 mg/kg, i.p.). The present study demonstrates that ethanol inhibits the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway by enhancing GABAA receptor-mediated neurotransmission.

Hyperresponsiveness of bronchial but not tracheal smooth muscle in a murine model of allergic bronchial asthma.

OBJECTIVE: To determine a change in airway smooth muscle contractility in a murine model of allergic asthma, the responsiveness of airway smooth muscles isolated from ovalbumin (OA)-sensitized and -challenged mice was compared with that from control animals. METHODS: Actively sensitized mice were repeatedly challenged by ovalbumin (OA) antigen inhalation. Twenty-four h after the last antigen challenge, tracheal and bronchial smooth muscle responsiveness to acetylcholine (ACh) and endothelin-1 (ET-1) were measured. Airway microvascular leakage and histochemistry were also determined as indices of airway inflammation. RESULTS: Both the ACh and ET-1 responsiveness of bronchial, but not tracheal, smooth muscles were significantly augmented in OA-challenged mice, whereas no significant change in the expression levels of M2, M3 and ETB receptors was observed. The Evans blue dye extravasation in the main bronchial, but not tracheal, tissue of OA-challenged mice was significantly increased as compared with that of sensitized control animals. A marked inflammatory cells infiltration was also observed in bronchial but not tracheal tissues of OA-challenged mice. CONCLUSION: Repeated antigen challenge to sensitized mice caused a hyperresponsiveness of bronchial, but not tracheal, smooth muscle accompanied with bronchial tissue inflammation.

Long-term outcome after allogeneic bone marrow transplantation for hematological malignancies with non-remission status. Results of a single-center study of 24 patients.

To investigate the problem of allogeneic bone marrow transplantation (allo-BMT) for advanced stage patients, we retrospectively analyzed 24 consecutive patients who underwent allo-BMT in the non-remission stage. Twenty-four patients (19 males and 5 females) with acute leukemia, chronic myelogenous leukemia, and malignant lymphoma underwent allo-BMT. The patients had a median age of 30 years. There were eight cases of acute myelogenous leukemia (AML), six cases acute lymphocytic leukemia (ALL), nine cases of chronic myelogenous leukemia (CML), and one case of Burkitt's lymphoma. The 3-year overall survival rate was 22.5%, with a median survival time of 206 days in AML, 345 days in ALL, and 363 days in CML. Overall survival was associated with a recovery of platelets of less than 30 days and an acute graft-versus-host disease (acute GVHD) presence of less than grade II ( p=0.042). Fourteen patients died of transplantation-related diseases. Our important problem is to decrease transplantation-related deaths in allo-BMT during the non-remission stage, and longer survival can be expected with better pretreatment and prophylaxis for GVHD. In addition, the selection of the source of hematopoietic stem cell transplantation at an optimal time is considered to be another problem to be approached.