Effects of heat-treated Lactobacillus crispatus KT-11 strain consumption on improvement of oral cavity environment: a randomised double-blind clinical trial.

Heat-treated lactic acid bacteria is added to some foods because it is easier to store and transport, and have less interference with other food ingredient compared with living lactic acid bacteria. We have reported that oral administration of heat-treated Lactobacillus crispatus KT-11 strain (KT-11) suppressed the symptoms of periodontal disease in mice orally infected with Porphyromonas gingivalis. However, the preventive effect of KT-11 on periodontal disease in human is unclear. The aim of this randomised double-blind clinical trial was to examine the effects of KT-11 consumption on the oral environment in healthy volunteers. Sixteen healthy volunteers were randomly assigned to two groups (KT-11 or placebo), and they consumed a KT-11 food tablet (1.2×1010 KT-11 cells) or a placebo food tablet (without KT-11) every day for 4 weeks. Clinical parameters including numbers of major periodontopathic bacteria in saliva, plaque score, gum conditions, and oral mucosal fluid level were evaluated at weeks 0 and 4. Amount of change in P. gingivalis numbers decreased in the KT-11 group compared to that in the placebo group at week 4. Female participants in the KT-11 group had decreased plaque scores, reddish tinge, and gingival swelling scores compared to those in the placebo group at week 4. Furthermore, male participants in the KT-11 group demonstrated increased oral mucosa fluid scores. These clinical findings suggest that daily KT-11 intake can prevent periodontal disease through the improvement of oral conditions.

A novel dual ex vivo lung perfusion technique improves immediate outcomes in an experimental model of lung transplantation.

The lungs are dually perfused by the pulmonary artery and the bronchial arteries. This study aimed to test the feasibility of dual-perfusion techniques with the bronchial artery circulation and pulmonary artery circulation synchronously perfused using ex vivo lung perfusion (EVLP) and evaluate the effects of dual-perfusion on posttransplant lung graft function. Using rat heart-lung blocks, we developed a dual-perfusion EVLP circuit (dual-EVLP), and compared cellular metabolism, expression of inflammatory mediators, and posttransplant graft function in lung allografts maintained with dual-EVLP, standard-EVLP, or cold static preservation. The microvasculature in lung grafts after transplant was objectively evaluated using microcomputed tomography angiography. Lung grafts subjected to dual-EVLP exhibited significantly better lung graft function with reduced proinflammatory profiles and more mitochondrial biogenesis, leading to better posttransplant function and compliance, as compared with standard-EVLP or static cold preservation. Interestingly, lung grafts maintained on dual-EVLP exhibited remarkably increased microvasculature and perfusion as compared with lungs maintained on standard-EVLP. Our results suggest that lung grafts can be perfused and preserved using dual-perfusion EVLP techniques that contribute to better graft function by reducing proinflammatory profiles and activating mitochondrial respiration. Dual-EVLP also yields better posttransplant graft function through increased microvasculature and better perfusion of the lung grafts after transplantation.

Biomechanical methods for the assessment of fracture repair.

The progress of fracture healing is directly related to an increasing stiffness and strength of the healing fracture. Similarly the weight bearing capacity of a bone directly relates to the mechanical stability of the fracture. Therefore, assessing the progress of fracture repair can be based on the measurement of the mechanical stability of the healing fracture. However, fracture stability is difficult to assess directly due to various obstacles of which shielding of the mechanical properties by the fracture fixation construct is the most relevant one. Several assessment methods have been proposed to overcome these obstacles and to obtain some sort of mechanical surrogate describing the stability of the fracture. The most direct method is the measurement of the flexibility of a fracture under a given external load, which comprises the challenge of accurately measuring the deformation of the bone. Alternative approaches include the measurement of load share between implant and bone by internal or by external sensors. A direct 3 dimensional measurement of bone displacement is provided by radiostereometric analysis which can assess fracture migration and can detect fracture movement under load. More indirect mechanical methods induce cyclic perturbations within the bone and measure the response as a function of healing time. At lower frequencies the perturbations are induced in the form of vibration and at higher frequencies in the form of ultrasonic waves. Both methods provide surrogates for the mechanical properties at the fracture site. Although biomechanical properties of a healing fracture provide a direct and clinically relevant measure for fracture healing, their application will in the near future be limited to clinical studies or research settings.

Effect of low-intensity pulsed ultrasound stimulation on callus remodelling in a gap-healing model: evaluation by bone morphometry using three-dimensional quantitative micro-CT.

We evaluated the effect of low-intensity pulsed ultrasound stimulation (LIPUS) on the remodelling of callus in a rabbit gap-healing model by bone morphometric analyses using three-dimensional quantitative micro-CT. A tibial osteotomy with a 2 mm gap was immobilised by rigid external fixation and LIPUS was applied using active translucent devices. A control group had sham inactive transducers applied. A region of interest of micro-CT was set at the centre of the osteotomy gap with a width of 1 mm. The morphometric parameters used for evaluation were the volume of mineralised callus (BV) and the volumetric bone mineral density of mineralised tissue (mBMD). The whole region of interest was measured and subdivided into three zones as follows: the periosteal callus zone (external), the medullary callus zone (endosteal) and the cortical gap zone (intercortical). The BV and mBMD were measured for each zone. In the endosteal area, there was a significant increase in the density of newly formed callus which was subsequently diminished by bone resorption that overwhelmed bone formation in this area as the intramedullary canal was restored. In the intercortical area, LIPUS was considered to enhance bone formation throughout the period of observation. These findings indicate that LIPUS could shorten the time required for remodelling and enhance the mineralisation of callus.

Control of fracture reduction robot using force/torque measurement.

We have developed a surgical robotic system for femoral fracture reduction employing indirect traction. Indirect traction in fracture reduction is a generally used surgical method for preventing complications such as bone splits caused by high stress on bones. For traction, a patient's foot is gripped by a jig and pulled to the distal side. Indirect traction has the advantage of distributing bone stress by utilizing a strong traction force; however, this procedure does not accurately control the proper positioning of fractured fragments when a surgical robot is used. The human leg has knee and an ankle joints, and thus robotic motion presents problems in not being able to directly propagate reduction motion to a fractured femoral fragment, rendering control of bone position difficult. We propose a control method for fracture reduction robots using external force/torque measurements of the human leg to achieve precise fracture reduction. Results showed that the proposed method reduced repositioning error from 6.8 mm and 15.9 degrees to 0.7 mm and 5.3 degrees, respectively.

Assessment of the pressure-volume relationship of the single ventricle of the grass shrimp, Palaemonetes pugio.

The ventricular pressure-volume (PV) relationship has been used extensively to study the mechanics and energetics in multi-chambered hearts of closed circulatory system vertebrates. In the current study we applied the use of PV loops in the assessment of cardiac mechanics and energetics in the single ventricle of a decapod crustacean possessing an open circulatory system. Anatomical differences between multi-and single-chambered hearts include multiple ostia entering and valved multiple arterial systems exiting the ventricle, and the neurogenic origin of the heartbeat in decapod crustaceans. However, the microscopic architecture and excitation-contraction coupling events are similar in both systems. Ventricular pressure and area were obtained independently and integrated into pressure-area loops. Area was then converted to volume to generate PV loops. Based on the PV loops generated in this study, the ventricle of Palaemonetes pugio processes the same primary phases of the cardiac cycle as ventricles from the multi-chambered hearts of vertebrates: (1) isovolumic contraction, (2) ventricular emptying, (3) isovolumic relaxation and (4) ventricular filling. The area enclosed by the PV loop provides a measure of stroke work and when multiplied by heart rate provides an assessment of cardiac work. This initial examination of PV loops from a single-ventricle decapod crustacean demonstrates the utility of this technique to further elucidate the cardiac mechanics and energetics of this system, and in particular during times of physiological stress.

Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production.

One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 x 10(9) genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.

Pancreatic acinar cell carcinoma with endocrine differentiation: immunohistochemical and ultrastructural analyses.

The majority of pancreatic malignant tumors are adenocarcinomas of the ductal type (ductal cell carcinomas) and combined tumors consisting of different tumor components are very rare. We present here a rare case of acinar cell carcinoma with apparent foci of endocrine differentiation. A 46-year-old man underwent pylorus-preserving pancreatoduodenectomy under the diagnosis of pancreatic tumor. The pancreatic tumor was mainly composed of typical acinar cell carcinoma, but some tumor cells were positive for both acinar and endocrine cell markers such as pancreatic amylase, trypsin, lipase and chromogranin A. At the electron-microscopic level, the tumor cells were seen to have numerous electron-dense neuroendocrine, as well as a few zymogen-like, granules. The tumor part positive for both acinar and endocrine cell markers originated from a subclone (dis-differentiated tumor cells) of the typical acinar cell carcinoma tissue of the pancreas.

MUC1 and MUC2 expression in human gallbladder carcinoma: a clinicopathological study and relationship with prognosis.

Mucins are a group of high molecular weight glycoproteins consisting of a mucin core protein and O-linked carbohydrates. To date, nine apomucins (MUC1-8, and MUC5B) have been identified. Recent studies have demonstrated that MUC1 is expressed in tumors of various human organs, and may function as an anti-adhesion molecule that inhibits cell-to-cell adhesion, inducing tumor metastasis. MUC2 is a major secreted mucin of colon and is known to be expressed in cells showing intestinal metaplasia in the stomach and other organs. MUC2 expression in the mucosal epithelia is an apparently abnormal phenomenon related to the neoplastic process. In this study, we examined MUC1 and MUC2 expression in human gallbladder adenocarcinoma and its clinicopathological significance and relationship with the prognosis of the patients. MUC1 immunoreactivity was detected not only in the cancer cells but also in the cancer stroma. Cytoplasmic MUC1 expression was significantly relation to lymphatic invasion and lymph node metastasis (p < 0.001), and was associated with a poor outcome. In contrast, MUC2 was rarely expressed in gallbladder carcinomas, and its immunoreactivity was detected only in the cancer goblet cells. Overexpression of MUC2 was not significantly related to lymphatic invasion or lymph node metastasis, or prognosis of patients. These observations suggested that MUC1 expression plays a more important role than MUC2 expression in cancer cell growth and metastasis of human gallbladder adenocarcinomas.

(S)-preferential detoxification of 4-hydroxy-2(E)-nonenal enantiomers by hepatic glutathione S-transferase isoforms in guinea-pigs and rats.

In guinea-pig liver cytosol, racemic 4-hydroxy-2(E)-nonenal (HNE), a reactive and highly toxic product released from biomembranes by lipid peroxidation, was detoxified (S)-preferentially by GSH conjugation mediated by glutathione S-transferases (GSTs) and (R)-preferentially by NAD(+)-dependent oxidation mediated by aldehyde dehydrogenase (ALDH). The GST-mediated detoxification of the HNE enantiomers proceeded at much higher rates than that mediated by ALDH in guinea-pig liver cytosol. All the major guinea-pig GSTs, A1-1, M1-1, M1-2 and M1-3*, isolated from guinea-pig liver cytosol also catalysed the (S)-preferential conjugation of the HNE enantiomers. The liver and other major tissues of guinea-pigs had no immunologically detectable level of a putative GSTA4-4 orthologue, which exists as a minor GST protein in rat, mouse and human livers and exhibits extremely high catalytic activity towards HNE. All the hepatic rat GSTs, A1-1(2), A1-3, A4-4, M1-1, M1-2 and M2-2, also catalysed the (S)-preferential conjugation of HNE enantiomers.

Solution properties of amino acid-type new surfactant.

The solution properties of an amino acid-type new surfactant, N-[3-lauryloxy-2-hydroxypropyl]-L-arginine L-glutamate (C12HEA-Glu), were examined by means of pK(a), surface tension, light scattering and fluorescence measurements. The dissociation state of C12HEA-Glu was changed by varying pH of the aqueous solution. The critical micelle concentration (cmc) of aqueous solution of C12HEA-Glu was lowest at pH 5.6 and the cmc increased with decreasing pH. The aggregation number of C12HEA-Glu micelles was largest at pH 5.6 and decreased with decreasing pH. The surface tension-concentration curve for C12HEA-Glu in water (non-buffered solution) showed a minimum. This is considered to be due to the coexistence of a trace species and a main species of C12HEA-Glu with various dissociation states since the pH of aqueous solution of C12HEA-Glu changes with the concentration of C12HEA-Glu. The micropolarity in the micelles of C12HEA-Glu was almost independent of pH, while the microfluidity in the micelles increased with increasing pH.

Self-amplification system for recombinant adeno-associated virus production.

A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins.

Right and left ventricular wall deformation patterns in normal and left heart hypoplasia chick embryos.

The vertebrate embryonic ventricle transforms from a smooth-walled single tube to trabeculated right ventricular (RV) and left ventricular (LV) chambers during cardiovascular morphogenesis. We hypothesized that ventricular contraction patterns change from globally isotropic to chamber-specific anisotropic patterns during normal morphogenesis and that these deformation patterns are influenced by experimentally altered mechanical load produced by chronic left atrial ligation (LAL). We measured epicardial RV and LV wall strains during normal development and left heart hypoplasia produced by LAL in Hamburger-Hamilton stage 21, 24, 27, and 31 chick embryos. Normal RV contracted isotropically until stage 24 and then contracted preferentially in the circumferential direction. Normal LV contracted isotropically at stage 21, preferentially in the longitudinal direction at stages 24 and 27, and then in the circumferential direction at stage 31. LAL altered both RV and LV strain patterns, accelerated the onset of preferential RV circumferential strain patterns, and abolished preferential LV longitudinal strain (P < 0.05 vs. normal). Mature patterns of anisotropic RV and LV deformation develop coincidentally with morphogenesis, and changes in these deformation patterns reflect altered cardiovascular function and/or morphogenesis.

Maturation of end-systolic stress-strain relations in chick embryonic myocardium.

The embryonic myocardium increases functional performance geometrically during cardiac morphogenesis. We investigated developmental changes in the in vivo end-systolic stress-strain relations of embryonic chick myocardium in stage 17, 21, and 24 white Leghorn chick embryos (n = 10 for each stage). End-systolic stress-strain relations were linear in all developmental stages. End-systolic strain decreased from 0.50 +/- 0.02 to 0.31 +/- 0.01 (mean +/- SE, P < 0.05), while average end-systolic wall stress was similar at 3.29 +/- 0.34 to 4.19 +/- 0.43 mmHg (P = 0.14) from stage 17 to 24. Normalized end-systolic myocardial stiffness, a load-independent index of ventricular contractility, increased from 2.98 +/- 0.19 to 6.03 +/- 0.39 mmHg from stage 17 to 24 (P < 0.05). Zero-stress midwall volume increased from 0.024 +/- 0.002 to 0.124 +/- 0.004 microl from stage 17 to 24 (P < 0.05). These results suggest that the embryonic ventricle increases normalized ventricular "contractility" while maintaining average end-systolic wall stress over a relatively narrow range during cardiovascular morphogenesis.

[X-ray and endoscopic diagnosis of esophageal achalasia].

We evaluated diagnostic X-ray and endoscopic examination findings in 486 patients with esophageal achalasia. Concerning the association between the duration of disease and the X-ray dilatation type, the duration was 1-4 years in more than 50% of patients with the Sp type, a mean of 8.5 years in those with the F type, and frequently more than 10 years in those with the S type. Endoscopy is generally used to determine the presence or absence of abnormal movement. In achalasia, the endoscope can be inserted into the stomach despite resistance at the stenotic site, and the mucosal surface is normal. Squamous cell carcinoma as a complication was observed in 21 patients (4.3%). The carcinoma complication rate was higher with a longer duration of disease and a longer observation period. The mean total course including the postoperative course was 27 years. Long-term and periodic X-ray and endoscopic observation of the disease course is important, and iodine staining is indispensable for early detection of esophageal cancer.

Stromal sialyl Le(a) expression is correlated with vascular invasion of human gallbladder adenocarcinoma.

Sialyl Le(a) antigen (CA19-9), a member of a family of high molecular weight glycoproteins, was originally described as a gastrointestinal- and pancreatic-specific tumor marker. Recent studies have demonstrated that sialyl Lea is a ligand for E-selectin and may play an important role in tumor metastasis. However, expression patterns of sialyl Le(a) have not yet been established in human gallbladder carcinomas. In this study, we examined sialyl Le(a) expression in human gallbladder adenocarcinoma and its clinicopathological significance. Sialyl Le(a) immunoreactivity was detected not only in cancer cells (cytoplasmic type; 68.5%, 37/54) but also in cancer stroma (stromal type; 46.3%, 24/54). According to TNM classification, stromal sialyl Le(a) expression was detected in 60. 0% (24/40) and 7.1% (1/14) of the T2-4 and T1 cancers, respectively (p<0.01). Stromal sialyl Le(a)-positive gallbladder cancers frequently showed lymphatic invasion, venous invasion and lymph node metastasis (62.9%, 62.5% and 70.0%, respectively) (p<0.01). These observations suggested that sialyl Le(a) expression plays important roles in vascular invasion and metastasis of human gallbladder adenocarcinomas.

DF3 expression in human gallbladder carcinoma: significance for lymphatic invasion.

DF3 (MUC 1) is a member of a family of high molecular weight glycoproteins. Recent studies have demonstrated that DF3 is expressed in tumors of various human organs, and may function as an anti-adhesion molecule that inhibits cell-to-cell adhesion, inducing tumor metastasis. However, expression patterns of DF3 have not yet been established in human gallbladder carcinomas. In this study, we examined DF3 expression in human gallbladder adenocarcinoma and its clinicopathological significance. DF3 immunoreactivity was detected not only in the cancer cells (cytoplasmic type; 50.0%, 27/54) but also in the cancer stroma (stromal type; 46.3%, 25/54). According to TNM classification, 65.0% (26/40) of T2-4 gallbladder cancers showed cytoplasmic DF3, while 7.1% (1/14) of the T1 cancers were cytoplasmic DF3-positive (p<0.001). Stromal DF3 expression was detected in 62.5% (25/40) and none (0/14) of the T2-4 and T1 cancers, respectively (p<0.001). Lymph node metastasis was frequently found in the cytoplasmic DF3- and stromal DF3-positive gallbladder cancers (59.3% and 60.0%, respectively). These observations suggested that DF3 expression plays important roles in cancer cell growth and metastasis of human gallbladder adenocarcinomas.

CEA immunohistochemical localization is correlated with growth and metastasis of human gallbladder carcinoma.

Carcinoembryonic antigen (CEA) is a good marker of colorectal cancer. Recent studies have demonstrated that CEA may function as a metastatic potentiator by different pathways; i.e., modulation of immune responses, facilitation of intercellular adhesion and cellular migration. However, expression patterns of CEA have not yet been established in human gallbladder carcinomas. In this study, we examined CEA expression in human gallbladder adenocarcinomas and its clinicopathological significance. CEA immunoreactivity was detected not only in the cancer cells (cytoplasmic type: 63.0%, 34/54) but also in the cancer stroma (stromal type: 29.6%, 16/54). According to TNM classification, 75.0% (30/40) of T2-4 gallbladder cancers showed cytoplasmic CEA, while 28.6% (4/14) of the T1 cancers were cytoplasmic CEA-positive (p<0.05). Stromal CEA expression was detected in 40.0% (16/40) and none (0/14) of the T2-4 and T1 cancers, respectively (p<0.05). Lymph node metastasis was frequently found in the cytoplasmic CEA- and stromal CEA-positive gallbladder cancers (44.1% and 62.5%, respectively). These observations suggested that CEA expression plays important roles in cancer cell growth and metastasis of human gallbladder adenocarcinomas.