Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3.

Effects of a flow field on amyloid fibrillogenesis in a ß-lactoglobulin solution.

The effects of a flow field on the amyloid fibrillogenesis of ß-lactoglobulin (ßLG) were investigated using a flow birefringence method and AFM imaging experiments. A 4 wt% ßLG aqueous solution was incubated at pH 2 and 80°C. A flow field was then applied by stirring at 250 and 474 rpm. An incubation without stirring was used as a control sample. Flow birefringence measurements were taken at room temperature from the incubated sample solutions in which an elongational flow field was used. The birefringence pattern obtained indicated that the fibrils formed by the incubation were rigid rod-like molecules. Birefringence relaxation experiments revealed at least two relaxation processes, suggesting a double peaked distribution function for fibrils length. The length distribution of fibrils expected from the birefringence experiments was confirmed by the AFM images of amyloid fibrils. The order of the expected length of the resultant fibrils in both longer and shorter length distributions was those stirred at 250 rpm≅474 rpm>0 rpm. The effects of the flow field applied during the incubation on amyloid fibrillogenesis was discussed on the basis of the rate process consideration. The present results demonstrated that the flow field should be considered as an important factor that regulates the fibrillogenesis of globular proteins.

Nanometric locking of the tight focus for optical microscopy and tip-enhanced microscopy.

We have successfully stabilized the tight focus onto the sample surface of an optical microscope within ±1.0 nm for a virtually unlimited time duration. The time-dependent thermal drift of the tight focus and the mechanical tilt of the sample surface were simultaneously sensed by a non-optical means based on a capacitive sensor and were compensated for in real-time. This non-optical scheme is promising for the suppression of background light sources for optical microscopy. The focus stabilization is crucial for microscopic measurement at an interface, particularly when scanning a large surface area, because there is always a certain amount of mechanical tilt of the sample substrate, which degrades the contrast of the image. When imaging nanoscopic materials such as carbon nanotubes or silicon nanowires, more stringent nanometric stabilization of the focus position relative to such samples is required, otherwise it is often difficult to interpret the results from the observations. Moreover, the smaller the sample volume is, the smaller the signal becomes, resulting in a long exposure time at each position. In this sense, long-term stability of the tight focus is essential for both microscopic large area scanning and nanosized sample scanning (high-resolution/large-area imaging). In addition, the recently developed tip-enhanced microscopy requires long-term stability of the relative position of the tip, sample and focus position. We were able to successfully demonstrate a stability improvement for tip-enhanced microscopy in the same manner. The stabilization of the tight focus enables us to perform long-term and robust measurements without any degradation of optical signal, resulting in the capability of true nanometric optical imaging with good reproducibility and high precision. The technique presented is a simple add-on for any kind of optical microscope.

Wheelchair half-marathon race increases natural killer cell activity in persons with cervical spinal cord injury.

STUDY DESIGN: Non-randomized study. OBJECTIVE: We reported that individuals with cervical spinal cord injury (CSCI) showed no increase in natural killer cell activity (NKCA) in response to 20-min arm exercise. It could be argued that this lack of response was owing to the short duration and intensity of the exercise. SETTING: The 29th Oita International wheelchair marathon race. METHODS: The present study compared the effects of wheelchair half-marathon race on natural killer (NK) cell count, NKCA and other hematological and hormonal parameters in six subjects with CSCI and seven control subjects with spinal cord injury between T4 and L1 (SCI), before, immediately after and 2 h after recovery. RESULTS: NK cell counts increased at both time points after the race in SCI, but not in CSCI, compared with before the race. NKCA increased immediately in both groups of subjects after the race, and then returned to the pre-race level at 2 h after the race. Plasma cortisol did not change in both groups throughout the study. Plasma adrenaline increased sharply in SCI after the race, then returned to the pre-race level at 2 h after the race, whereas no change was observed in CSCI throughout the study. CONCLUSION: The present study demonstrated that wheelchair half-marathon race increases NKCA despite the lack of increase in plasma adrenaline in CSCI, suggesting the activation of NKCA by mechanisms other than circulating adrenaline level.

Effect of age on bowel management in traumatic central cord syndrome.

STUDY DESIGN: A retrospective multicenter study. OBJECTIVES: To investigate the characteristics of bowel dysfunction in elderly people with traumatic central cord syndrome (TCCS). SETTING: A total of 28 Rosai hospitals in Japan. METHODS: The Rosai Hospital registry included 3006 persons with spinal cord injury during 1997-2007. The study subjects were 186 patients with TCCS (160 men, 26 women; mean age, 61.7±11.6 years, ±s.d.). Patients were divided according to age into the young group (<50 years, n=30), the middle-age group (50-69 years, n=112) and the elderly group (≥70 years, n=44). We assessed the differences in bowel management techniques (spontaneous, rectal medications and manual emptying) and activity of daily living (ADL) with respect to bowel care at discharge among the three groups. RESULTS: Continent spontaneous defecation was the most common bowel management method (50%, 93/186). The percentage of elderly subjects on continent spontaneous defecation (36.4%) was significantly less than that of the young group (66.7%; P<0.05). Furthermore, the percentage of elderly patients who required no bowel care (18.2%) was significantly less than those of the young (53.3%) and middle-age groups (41.1%; P<0.01). However, few differences in bowel care-related ADL were recognized among the three groups in patients who required manual emptying. CONCLUSION: The results identified significantly fewer patients aged ≥70 years with 'continent spontaneous defecation' or 'independent for bowel care' compared with younger patients. The results also highlighted the clinical importance of bowel dysfunction associated with TCCS especially in elderly people.

Plasma IL-6 levels during arm exercise in persons with spinal cord injury.

STUDY DESIGN: Non-randomized study. OBJECTIVES: Previous studies indicated that at least 2-h leg exercise at more than 60% maximum oxygen consumption (VO(2)max) increased plasma interleukin (IL)-6 in able-bodied (AB) subjects. The purpose of the present study was to compare IL-6 response to arm exercise in AB subjects and persons with spinal cord injury (SCI). SETTING: Wakayama Medical University in Japan. METHODS: Six subjects with SCI between T6 and T10 and seven AB subjects performed 2-h arm crank ergometer exercise at 60%VO(2)max. Plasma catecholamines, IL-6, tumor necrosis factor (TNF)-α and high-sensitivity C-reactive protein (hsCRP) were measured before exercise, 60-min exercise, immediately and 2 h after the completion of exercise. RESULTS: Arm exercise increased myoglobin and plasma IL-6 levels in SCI and AB (P<0.01), but there were no differences in them between the two groups throughout the study. Plasma levels creatine kinase, lactate dehydrogenase, TNF-α and hsCRP did not change throughout the study in both groups. CONCLUSION: These findings suggest neither significant muscle damage nor inflammatory response during exercise. The increase in plasma IL-6 in SCI was not unexpected, confirming that moderate intensity and relatively long-arm exercise is safe and beneficial for SCI subjects with regard to IL-6 excretion, as in AB subjects.

Incidence of symptomatic autonomic dysreflexia varies according to the bowel and bladder management techniques in patients with spinal cord injury.

STUDY DESIGN: A retrospective, multicenter study. OBJECTIVES: To investigate the relationship between bowel and bladder management methods and symptomatic autonomic dysreflexia (AD) during hospitalization in patients with spinal cord injury (SCI). SETTING: Twenty-eight Rosai hospitals in Japan. METHODS: The study subjects were 571 patients with SCI who had been admitted to 28 Rosai hospitals between April 1997 and March 2007 for rehabilitation therapy and fulfilled the following criteria: (1) SCI at or above sixth thoracic level, (2) discharged from hospital after more than 4 months of admission for initial injury and (3) lack of pressure ulcers, deep venous thrombosis, ureteral and renal stones or heterotopic ossification throughout hospitalization to exclude possible influence of these complications on cardiovascular reflexes. The study subjects were examined for the incidence of symptomatic AD according to age, sex, ASIA Impairment Scale, injury level, bowel and bladder management techniques at discharge. RESULTS: The Rosai Hospital registry included 3006 persons with SCI during 1997-2007, and 571 patients fulfilled the above criteria. The highest incidence of symptomatic AD was diagnosed in subjects using reflex voiding and in those using manual removal of stool. By contrast, the lowest incidence of symptomatic AD was in those on continent spontaneous voiding and continent spontaneous defecation. CONCLUSION: Medical staff should evaluate the presence of AD in patients with SCI at or above the T6 level under bladder and bowel management such as reflex voiding and manual removal of stool.

Effect of pH on anisotropic gelation of DNA induced by aluminum cations.

To investigate the effects of pH on the structure and the properties of DNA anisotropic gels and their growth process, we have observed the morphology of DNA anisotropic gel films prepared from DNA solutions with various initial pH, and measured the pH dependences of the shrinking ratio, the birefringence, and the relaxation modulus of the gel as well as the time courses of the gel front and pH-change front lines. The gel films prepared from DNA solutions with high pH have inhomogeneous macroscopic structure, large shrinking ratio, and high optical anisotropy whereas those prepared from DNA solutions with low pH have homogeneous macroscopic structure, small shrinking ratio, and low optical anisotropy. The difference observed at different pH is attributed to the difference in the interaction between DNA molecules and aluminum cations. The time courses of the gel front and pH-change front lines were analyzed with theories based on assumptions for each condition. Both two-stage dynamics observed under high initial pH and one-stage dynamics under low initial pH were explained consistently with the theories.

Impaired immune response to voluntary arm-crank ergometer exercise in patients with cervical spinal cord injury.

STUDY DESIGN: Non-randomized study. OBJECTIVE: The mechanism underlying exercise-induced argumentation of natural killer cell cytotoxic activity (NKCA) in humans remains unclear. To address this, NKCA responses were studied during and after exercise in persons with cervical spinal cord injury (CSCI) and dysfunctional sympathetic nervous system. SETTING: Kibikogen Rehabilitation Center for Employment Injuries. METHODS: We examined the NKCA responses to 20-min arm-crank ergometer exercise at 60% of maximum oxygen consumption in eight persons with CSCI (between C6 and C7) and six able-bodied subjects. NKCA, adrenaline, and cortisol were measured before, immediately after exercise, 1 h after exercise, and 2 h after exercise. RESULTS: In able-bodied subjects, NKCA increased immediately after exercise (P<0.01) and then decreased to below the pre-exercise level 1 h after exercise, before recovering to the baseline level at 2 h after exercise. Plasma adrenaline concentrations increased significantly immediately after exercise (P<0.01) and returned to the baseline level 1 h after exercise. The plasma cortisol level did not change throughout the study. In contrast, NKCA, plasma concentrations of adrenaline, and cortisol did not change throughout the study in subjects with CSCI. CONCLUSION: In subjects with CSCI, the lack of response in NKCA throughout the experiment is probably mainly due to a dysfunctional sympathetic nervous system.

Inorganic polyphosphate induces osteoblastic differentiation.

Inorganic polyphosphate [Poly(P)] is especially prevalent in osteoblasts. We tested the hypothesis that Poly(P) stimulates osteoblastic differentiation and polyphosphate metabolism for bone formation. The osteoblast-like cell line, MC 3T3-E1, was cultured with Poly(P), and gene expression was evaluated by real-time reverse-transcription polymerase chain-reaction. Phosphatase activity and extracellular matrix mineralization were also determined. The role of Poly(P) was assessed in a beagle dog alveolar bone regeneration model. Poly(P) increased osteocalcin, osterix, bone sialoprotein, and tissue non-specific alkaline phosphatase gene expression, with a high level of end-polyphosphatase activity, resulting in low-chain-length Poly(P), inorganic pyrophosphate, and inorganic phosphate production. MC3T3-E1 cells differentiated into mature osteoblasts and showed expression of ectonucleotide pyrophosphatase phosphodiesterase 1, while mouse progressive ankylosis gene expression remained unchanged. Promotion of alveolar bone regeneration was observed in Poly(P)-treated beagle dogs. These findings suggest that Poly(P) induces osteoblastic differentiation and bone mineralization, and acts as a resource for mineralization.

Topical anesthesia blunts the pressor response induced by bowel manipulation in subjects with cervical spinal cord injury.

DESIGN: Prospective double-blind study. OBJECTIVE: Manual emptying of rectal contents is necessary for patients with spinal cord injury to prevent bowel obstruction; however, this maneuver induces a rise in blood pressure (BP) and autonomic dysreflexia (AD). The purpose of this prospective double-blind study was to investigate whether topical anorectal anesthesia attenuates the BP rise and AD during bowel manipulation in patients with cervical spinal cord injury (CSCI). SETTING: Kibikogen Rehabilitation Center for Employment Injuries. METHODS: The study subjects were 25 consecutive clinically stable patients with CSCI. Each subject received a complete bowel program involving manual removal of stool in lateral recumbency, after topical application of lidocaine or placebo jelly to the anorectal area. Systolic and diastolic BP, heart rate and symptoms of AD were recorded before, during and after bowel manipulation. RESULTS: Systolic BP was significantly lower during the lidocaine arm of the study compared with placebo at insertion of rectal medication, digital stimulation, beginning of stool flow, manual removal of stool, end of stool flow and at 5-min after emptying. The mean maximal increase in systolic BP during lidocaine treatment (33.2+/-14.6 mm Hg) was less than during placebo (50.2+/-19.5 mm Hg, P<0.001). CONCLUSION: On the basis of our findings, we recommend induction of lidocaine jelly immediately before rectal manipulation in patients with CSCI to minimize the incidence and severity of AD.

Attenuation of natural killer cell activity during 2-h exercise in individuals with spinal cord injuries.

DESIGN: Non-randomized study. OBJECTIVE: To determine natural killer cell cytotoxic activity (NKCA) to 2-h arm ergometer exercise in persons with spinal cord injuries (SCI) and the underlying mechanism of such response. SETTING: University of Occupational and Environmental Health, Japan. METHODS: We examined NKCA response to 2-h arm crank ergometer exercise at 60% of maximum oxygen consumption (VO(2max)) in SCI and able-bodied persons. NKCA and plasma concentrations of prostaglandin E(2) (PGE(2)), adrenaline and cortisol were measured before, during and immediately after the exercise. The study included seven subjects with SCI between Th11 and L4 and six able-bodied persons. RESULTS: NKCA in able-bodied subjects increased (P<0.05) at 60 min of exercise and immediately after the exercise, and remained elevated up to 2 h after exercise. However, NKCA in SCI decreased (P<0.05) immediately after exercise but recovered at 2 h after exercise. Plasma adrenaline in both groups increased significantly (P<0.05) immediately after exercise and returned to baseline level 2 h after the exercise. Plasma cortisol in both groups remained constant throughout the study. In SCI, PGE(2) significantly increased immediately after 2 h exercise and returned to the baseline level 2 h after exercise; however, it remained unchanged during the test in able-bodied subjects. CONCLUSION: Our results suggested that increase of PGE(2) in SCI partially contributes to NKCA.

Dynamics of liquid crystalline gelation of DNA.

DNA liquid crystalline gel (LCG) films have been prepared by immersing DNA aqueous borate solutions sandwiched between two circular glass plates into cobalt chloride solutions. The time courses of the thickness, the weight fractions of DNA and cobalt cations, and the birefringence and turbidity of the film consisted of outer DNA LCG and inner DNA amorphous gel or solution have been measured. To clarify the mechanism of the process forming LCG, the theory based on the nonequilibrium thermodynamics with "moving boundary picture" [Langmuir 2005, 21, 8155-8160] was modified, and the results were analyzed by the modified theory. It was found that the growth process of DNA LCG consists of two dynamics: cobalt cation diffusion-limited process at the early stage and the DNA circumstance change limited process at the later stage.

Inorganic polyphosphate: a possible stimulant of bone formation.

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

The incidence of post-race symptoms of upper respiratory tract infection in wheelchair marathon racers.

STUDY DESIGN: Prospective analysis. OBJECTIVES: To investigate the influence of exercise and major competition on infectious episodes in athletes with spinal cord injuries (SCI). SETTING: Japan. METHODS: We examined the self-reported infectious episodes of upper respiratory tract infection (URTI) in athletes with SCI during a 1-month period before the race and 2 weeks after the race. The study included 21 persons with SCI who participated in the 18th Oita International Wheelchair Marathon. Thirteen persons with SCI who did not participate in the race were studied as control subjects. RESULTS: The number of URTI episodes in marathoners was 0.086+/-0.036/week during the 1-month period before the race and 0.089+/-0.040/week during the 2-week post-race period, whereas that of the controls was 0.139+/-0.046/week during the 1-month period before the race and 0.072+/-0.047/week during the 2-week post-race period. There were no significant differences between before and after the race in marathoners, or between marathoners and controls during each period. However, the number of URTI episodes 2 weeks after the race was significantly higher in subjects who trained more than 65 km/week compared to those who trained less than 65 km/week. CONCLUSIONS: In subjects with SCI who completed a wheelchair full marathon race, the incidence of URTI after the race was not high compared to control subjects who did not participate in the race. According to the number of URTI episodes and the training data, it is recommended that wheelchair marathoners should consider their risk for URTI during excessive practice.

Tissue reaction to poly (lactic-co-glycolic acid) copolymer membrane in rhBMP used rabbit experimental mandibular reconstruction.

A rabbit experimental mandibular defect was reconstructed with 1% atelocollagen gel including rhBMP-2 10microg and a covering a poly (lactic-co-glycolic acid) copolymer (PLGA) membrane. For this experiment, eight male rabbits were used and a histological study was conducted. Our study purpose was to examine the effects and fate of PLGA membrane during bone reconstruction. PLGA membrane was phagocytized by foreign body giant cells and macrophages in the healing course of reconstruction osteogenesis. These histological data suggest that the PLGA membrane was gradually absorbed and replaced by fibrous connective tissue or bone tissue. In the osteogenesis course, the outer periphery of the new bone was maintained by PLGA membrane without expansion.

Three-dimensional observation of reconstruction course of rabbit experimental mandibular defect with rhBMP-2 and atelocollagen gel.

For the experimental animals, eight rabbits were chosen. A bone defect was made and was filled with 1% atelocollagen gel including rhBMP-2 10 microg. The reconstruction course was observed using micro-computed tomography (muCT) in vivo. In muCT observation, the density was slightly elevated at the bone marrow side at day 7, and the phenomenon gradually expanded during the course of this experiment which lasted for 28 days. By utilized muCT, we could construct 3D images, and that process enabled us to visualize bone formation more closely. These data suggest that the experimental animal model muCT and 3D image are extremely useful for follow-up of reconstruction of animal bone defects and that the atelocollagen gel is effective as a carrier of rhBMP-2.

Bone repair of rabbit mandibular transsection using rhBMP-2 and atelocollagen gel.

This experimental was carried out using 12 rabbits. A rabbit experimental mandibular transsection was reconstructed with 10mug of rhBMP-2 and 1% atelocollagen gel. The transsection gap was fixed with a titanium plate and screws, and covered with poly (lactic-co-glycolic acid) co-polymer (PLGA) membrane. Histopathological examination of 1-week specimens revealed that many spindle cells had proliferated and invaded blood clots, and a small amount of immature trabecular bone was formed in the transsection gap. In 2 and 3-week specimens, bone formation was gradually increased in the fibro-vascular tissues of the site. These histological findings were also observed in the control group specimens, but the bone formation was slightly less than in the experimental group. The results suggest the effectiveness of atelocollagen gel as a carrier of rhBMP-2 and PLGA as a covering membrane in this rabbit mandibular transsection reconstruction model.

Brillouin characterization of holey optical fibers.

We report the results of detailed measurements on the Brillouin frequency shift (BFS), gain bandwidth, and gain coefficients of several small-core holey optical fibers (HFs) of both uniform and axially varying structural characteristics and compare these with measurements on more conventional fibers. Our measurements show that the BFS of HFs is first-order proportional to the modal index for light propagating along the fiber and is thus extremely sensitive to its precise structural parameters. Our results highlight the possibility of using distributed Brillouin scattering measurements to perform nondestructive structural characterization of HFs, and the possibility of producing Brillouin-suppressed HFs using controlled structural variation along the fiber length.

Overeruption without root exposure of third molars and periodontal health in the mandible.

Bone formation is seen around the third molar even when the tooth is exposed to the oral environment due to overeruption. To determine if overeruption of the third molar with or without root exposure is related to the status of the exposure of other teeth in the mandible, using orthopantomographs, 424 third molars were studied in 371 patients who were over 41 years of age. The rate of overeruption and root exposure in third molars was measured, and its relationship to the number of teeth lost and the rate of root exposure in other teeth in the mandible was analyzed. Tooth loss in the group of third molars with overeruption without root exposure was greater than in that without overeruption or root exposure in men, whereas the relationship was not seen in women. We found that root exposures of other teeth in the group of third molars with overeruption without root exposure were significantly smaller than in those with root exposure in both genders. Third molars with overeruption without root exposure, in which bone formation was easy to observe for radiographic diagnosis, were correlated with periodontal health in the mandible, suggesting a component of precision determination for predicting resistance to periodontitis.