Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium WST-1.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric microbial viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7-14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3-4 days using the WST-1 colorimetric method and those obtained after 10-14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.

Self-monitoring has potential for home exercise programmes in patients with haemophilia.

Haemophiliacs who have had to keep a physically inactive lifestyle due to bleeding during childhood are likely to have little motivation for exercise. The purpose of this study is to clarify the effectiveness of the self-monitoring of home exercise for haemophiliacs. A randomized controlled trial was conducted with intervention over 8 weeks at four hospitals in Japan. Subjects included 32 male outpatients aged 26-64 years without an inhibitor who were randomly allocated to a self-monitoring group and a control group. Individual exercise guidance with physical activity for improvement of their knee functions was given to both groups. The self-monitoring materials included an activity monitor and a feedback system so that the self-monitoring group could send feedback via the Internet and cellular phone. The self-monitoring was performed by checking exercise adherence and physical activity levels, bleeding history and injection of a coagulation factor. Both groups showed significant improvements in exercise adherence (P < 0.001) and physical function such as the strength of knee extension (P < 0.001), range of knee extension (P < 0.001), range of ankle dorsiflexion (P < 0.01), a modified Functional Reach (P < 0.05) and 10 metre gait time (P < 0.01). In particular, improvements in exercise adherence (P < 0.05), self-efficacy (P < 0.05), and strength of knee extension (P < 0.05) were significant in the self-monitoring group compared with those in the control group. No increase in bleeding frequency and pain scale was noted. The self-monitoring of home exercise for haemophilic patients is useful for the improvement of exercise adherence, self-efficacy and knee extension strength.

Fine structural and immunohistochemical detection of collar enamel in the teeth of Polypterus senegalus, an actinopterygian fish.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 µm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

Changes of human menisci in osteoarthritic knee joints.

OBJECTIVE: To investigate the changes of knee menisci in osteoarthritis (OA) in human. METHODS: OA and control menisci were obtained from 42 end-stage OA knees with medial involvement and 28 non-arthritic knees of age-matched donors, respectively. The change of menisci in OA was evaluated by histology, and gene expression of major matrix components and anabolic factors was analyzed in the anterior horn segments by quantitative PCR (qPCR). In those regions of menisci, the rate of collagen neo-synthesis was evaluated by [(3)H]proline incorporation, and the change of matrix was investigated by ultrastructural observation and biomechanical measurement. RESULTS: In OA menisci, the change in histology was rather moderate in the anterior horn segments. However, despite the modest change in histology, the expression of type I, II, III procollagens was dramatically increased in those regions. The expression of insulin-like growth factor 1 (IGF-1) was markedly enhanced in OA menisci, which was considered to be responsible, at least partly, for the increase in procollagen gene expression. Interestingly, in spite of marked increase in procollagen gene expression, incorporation of [(3)H]proline increased only modestly in OA menisci, and impaired collagen synthesis was suggested. This finding was consistent with the results of ultrastructural observation and biomechanical measurement, which indicated that the change of meniscal matrix was modest in the macroscopically preserved areas of OA menisci. CONCLUSION: Although the expression of major matrix components was markedly enhanced, matrix synthesis was enhanced only modestly, and the changes of matrix in human OA menisci were rather modest in the non-degenerated areas.

Pedicle involvement on MR imaging is common in osteoporotic compression fractures.

BACKGROUND AND PURPOSE: Pedicle involvement on MR imaging has been considered specific for malignancy. However, we also noted the findings in many patients with osteoporosis and hypothesized that it is not specific for malignant lesions. The aim of this study was to evaluate the prevalence of pedicle involvement in painful osteoporotic compression fractures and to determine whether the sign is specific for malignancy. MATERIALS AND METHODS: We retrospectively reviewed MR images and CT scans of 152 patients who underwent PV for painful compression fractures. There were 140 patients (225 vertebrae) with osteoporotic fractures and 12 patients (19 vertebrae) with malignant fractures. Three radiologists evaluated the degree and extent of signal-intensity changes of the pedicle on MR imaging by consensus. The CT findings were also evaluated. The chi(2) test was used for statistical analyses. RESULTS: Of the 225 vertebrae of osteoporotic fractures and 19 vertebrae of malignant fractures, pedicle involvement on MR imaging was seen in 144 (64%) and 16 (84.2%) vertebrae, respectively, and there was no statistically significant difference (P = .065). Positive pedicle involvement in osteoporotic fractures was seen in 84 (77%) of 109 vertebrae with early-phase fractures (< or =3 months) and 60 (51.7%) of 116 vertebrae with chronic-phase fractures (>3 months), and this was statistically significant (P < .001). Among 144 osteoporotic vertebrae that showed positive pedicle involvement on MR imaging, 45 (31%) showed pedicle fractures and 55 (38.2%) showed sclerotic change on CT. CONCLUSIONS: Pedicle involvement was seen frequently in patients with osteoporotic compression fractures and was not specific for malignancy in our study group.

[Ross procedure with aortic replacement for aortic stenosis with ascending aortic aneurysm: report of a case].

Bicuspid aortic valve (BAV) is a common congenital heart disease, and it is well known to be a risk factor for ascending aortic dilatation and dissection. We here report a case of 34-year-old woman who underwent Ross procedure with ascending aortic replacement under the diagnosis of subaortic stenosis and ascending aortic aneurysm. She was pointed out to have heart murmur soon after the birth diagnosed as patent ductus arteriosus. The ductus was ligated when she was 3-years-old, however, heart murmur remained. Further examinations revealed that she also had aortic stenosis with BAV. During her 20-year-follow-up, subaortic stenosis and ascending aorta ectasia were also progressed. Pathological examinations of resected ascending aortic wall showed mucoid degeneration and laceration of collagen fibers, suggesting the fragility of dilated aortic wall with BAV.

Validation of amelogenesis imperfecta inferred from amelogenin evolution.

We used the evolutionary analysis of amelogenin (AMEL) in 80 amniotes (52 mammalian and 28 reptilian sequences) to aid in the genetic diagnosis of X-linked amelogenesis imperfecta (AIH1). Out of 191 residues, 77 were found to be unchanged in mammals, and only 34 in amniotes. The latter are considered crucial residues for enamel formation, while the 43 residues conserved only in mammals could indicate that they play new, important roles for enamel formation in this lineage. The 5 substitutions leading to AIH1 were validated when the mammalian dataset was used, and 4 of them with the amniote dataset. These 2 sequence datasets will facilitate the validation of any human AMEL mutation suspected of involvement in AIH1. This evolutionary analysis also revealed numerous residues that appeared to be important for correct AMEL function, but their role remains to be elucidated.

Brachial-ankle pulse wave velocity measured automatically by oscillometric method is elevated in diabetic patients with incipient nephropathy.

AIMS: To examine whether brachial-ankle pulse wave velocity (baPWV), a possible early marker of atherosclerotic vascular damage, is associated with albuminuria in patients with Type 2 diabetes. METHODS: BaPWV was measured by automatic oscillometric method in 346 Type 2 diabetic patients with normoalbuminuria (a mean level of three times measurements of albumin-to-creatinine (ACR)<30 microg/mg creatinine; n=200), incipient nephropathy (a mean level of ACR> or =30 and <300 microg/mg creatinine; n=119), and clinical nephropathy (a mean level of ACR> or =300 microg/mg creatinine; n=27), and without peripheral vascular disease. RESULTS: BaPWV (cm/s) was significantly higher in patients with incipient nephropathy (1722 +/- 382) and clinical nephropathy (1763 +/- 322) than in patients with normoalbuminuria (1559 +/- 343, P<0.0001, respectively). By univariate analysis it correlated significantly with age (r=0.44, P<0.0001), systolic blood pressure (r=0.55, P<0.0001), diastolic blood pressure (r=0.42, P<0.0001), albuminuria (r=0.24, P<0.0001) and HbA1C (r=0.11, P<0.05). Albuminuria revealed an independent significant association with baPWV (P<0.01) after adjustment for age, sex, smoking, BMI, HbA1C, hyperlipidemia, and hypertension. Multiple regression analysis showed age, diastolic blood pressure and albuminuria were independently associated with baPWV (adjusted R2=0.42, P<0.0001). CONCLUSIONS: The results might indicate a possible link between the pathogenesis of atherosclerosis and diabetic nephropathy. Future studies are needed to clarify the usefulness and its predictable value.

[A case report of ross operation and ventricular septal defect closure following correction of type A interruption by modified Blalock-Park, pulmonary artery banding and patent ductus arteriosus division].

A 2-year-old boy who had undergone a correction of a type A interruption using a modified Blalock-Park operation, pulmonary artery banding and the division of a patent ductus arteriosus, underwent a Ross operation and closure of ventricular septal defect (VSD). Although a pre-operative echo cardiogram revealed a bicuspid aortic valve, and a Doppler echocardiogram showed only 10 mmHg of pressure gradient across the aortic valve, Ross procedure was performed as a procedure accompanying the closure of a total conus VSD. The total conus VSD was closed with a Dacron patch using pledget mattress sutures. In addition, a running suture was applied over the denuded aortic root and the cranial margin to achieve water tight closure. An aortic root replacement procedure was our first choice for the Ross operation. After both coronary buttons were re-implanted into pulmonary sinuses, a pulmonary artery autograft was wrapped around by the remaining aortic wall for reinforcement to prevent future dilatation. The main pulmonary artery was reconstructed using a bicuspid pericardial valve conduit with a diameter of 24 mm. A post-operative echocardiogram showed no neoaortic valve regurgitation, good coaptation of tri-leaflets, mild regurgitation of pericardial valve and good cardiac performance.

[A modified Fontan operation in the presence of a supracardiac total anomalous pulmonary venous connection].

We report a case of 2-year-old girl with asplenia syndrome who successfully underwent modified Fontan procedure and concomitant repair of supracardiac total anomalous pulmonary venous connection (TAPVC). The preoperative diagnosis included a common atrioventricular canal (type C), a double outlet right ventricle, a common atrium, common atrioventricular valve regurgitation, pulmonary stenosis, and a bilateral superior vena cava (SVC). Cardiac catheterization revealed a Qp/Qs of 1.3, mean PA pressure of 16 mmHg and an Rp of 1.3. The TAPVC drained to left SVC (LSVC) at a position proximal to the hemiazygos vein with an ostium of 5 mm in diameter. The LSVC was divided distal to its connection to the common pulmonary vein (CPV). The TAPVC ostium was cut back into the CPV, then it was anastmosed with posterior aspect of the atrial wall in an effort to provide a wide anastomosis. The postoperative course was uneventful and the patient was discharged from hospital on the 35th postoperative day.

Conservative treatment of hemolytic complication following coil embolization in two adult cases of patent ductus arteriosus.

Two adult cases of relatively large patent ductus arteriosus (PDA) were treated by coil embolization, but were complicated by hemolysis that was successfully managed by medical treatment. Case 1 was a 67-year-old woman and Case 2 was a 71-year-old woman with a PDA of minimal diameter of 5.3 mm and 5.5 mm, respectively. The approach was via the pulmonary artery and 2 coils were delivered simultaneously into the ductus, known as the 'kissing coil technique'. Although immediately after the procedure only a small residual shunt was revealed by aortogram, hemolysis occurred for several hours after the procedure in both cases. A hemolytic complication usually needs additional coil embolization or surgical treatment, but in these 2 cases it was successfully treated by haptoglobin infusion to prevent nephropathy and by antiplasmin infusion to promote thrombus formation. Hemolytic complications of coil embolization of PDA can managed by medication when the residual shunt is minimal and the degree of hemolysis is mild.

Abortive secretion of an enamel matrix in the inner enamel epithelial cells during an enameloid formation in the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii).

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

Villiform processes in the pharynx of the soft-shelled turtle, Trionyx sinensis japonicus, functioning as a respiratory and presumably salt uptaking organ in the water.

Some species of soft-shelled turtle have been known to use a conspicuous mass of villiform processes of the pharyngeal mucosa as an aquatic respiratory organ when staying underwater for prolonged periods, such as hibernation. Using hibernating turtles, Trionyx sinensis japonicus, the present study employed scanning electron microscopy to demonstrate for the first time the detailed morphology and distribution of these villiform processes. Two types of processes, complex and simple, could be identified. Light microscope observation of the transverse sections of the villi demonstrated a rich vascularization in the connective tissue of the villi, comprising arterioles and venules running in the core and capillaries in the periphery. Most of the capillaries were invaginated into the multilayered cuboidal epithelium. Near the tip of the villi they became swollen, forming sinusoidal capillaries. Transmission electron microscopy clarified the fine structure of the blood-water barrier, which consisted of a non-fenestrated endothelium and an attenuated epithelium that sandwiched a connective tissue with a discontinuous subendothelial and a continuous subepithelial basement lamina. The epithelium consisted of secretory cells, mitochondria-rich cells, and basal cells. The mitochondria-rich cells contained a cytoplasmic area filled with tubulovesicular elements. Based on their ultrastructural resemblance with the chloride cells in the fish and tadpole, these cells are suggested to be involved in the uptake of Na+ and Cl from fresh water for keeping ionic balance in the blood.

The vascular supply of the villiform processes in the pharynx of the soft-shelled turtle, Trionyx sinensis japonicus. A scanning electron microscopic study of corrosion casts.

Following our observations of the fine structure of the pharyngeal villiform processes of the hibernating soft-shelled turtle, Trionyx sinensis japonicus (Yokosuka et al., 2000), this paper deals with a scanning electron microscope study of the resin casts of blood vessels supplying those processes. Each villiform process contained arterioles and venules which ran in the axial portion of the process; capillaries formed a network at the periphery of the connective tissue core of the villus. In the distal portions of the villus, the capillaries increased markedly in their caliber to form sinusoidal capillaries. Such a vascular architecture supports the view that the villiform processes serve in the aquatic respiration of the soft-shelled turtle. The casts indicated an occurrence of sphincters in the vascular bed of the villi.

An immunocytochemical study of amelogenin proteins in the developing tooth enamel of the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii).

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

Physical and mental correlates of hearing impairment in the elderly in Japan.

Seven hundred and forty seven elderly people aged from 65 to 98 (209 male and 538 female) admitted to the Geriatric Health Service Facility were evaluated with pure-tone audiometry, Office of Population Censuses and Surveys (OPCS) personal care severity score, OPCS hearing severity score, Mini-mental State Examination (MMSE) and Self-rating Depression Scale (SDS). A single correlation study indicated that the hearing level significantly correlated with age, OPCS hearing severity score, MMSE total score and SDS total score. A multiple regression analysis showed that age, OPCS hearing severity score, and MMSE total score were significantly associated with the hearing level. We demonstrated here that the objective auditory function strongly correlated with the cognitive function measured by MMSE total score, suggesting that a regular audiological check-up and the early detection of the hearing impairment could contribute to the maintenance of the quality of life in the elderly.

Infant cold exposure changes Fos expression to acute cold stimulation in adult hypothalamic brain regions.

It is known that cold exposure as an infant results in a permanent cold tolerance which is accompanied by a reduced rate of colonic temperature decline and increased metabolic heat production to cold stimulation. The present study was aimed to elucidate the central mechanism of cold tolerance of adult rats with cold exposure as infants. Newborn infants were exposed to cold ambient (4 degrees C) daily for 2 weeks, and when they were 15-weeks old, Fos expression to acute cold stimulation (10 degrees C) in whole brain regions was investigated using quantitative immunohistochemistry. Acute cold stimulation induced a significant increase of Fos-positive neurons in many nuclei of whole brain areas in adult rats both with and without cold exposure as infants. However, the number of Fos-positive neurons was significantly less in the rats with cold exposure than without cold exposure as infants, particularly in the hypothalamic nuclei such as the lateral septal nucleus (LS), preoptic area (POA), parvocellular paraventricular nucleus (pPVN0, ventromedial hypothalamic nucleus (VMH) and supramammillary nucleus (SuM). However, there was no significant difference in the number of Fos-positive neurons in the extra-hypothalamic regions between the rats with and without cold exposure as infants. These results suggest that the central mechanism of cold tolerance with cold exposure as infants is due to metabolic changes of the hypothalamic brain regions.

Amelogenin protein in tooth germs of the snake Elaphe quadrivirgata, immunohistochemistry, cloning and cDNA sequence.

In the snake, Elaphe quadrivirgata, the occurrence of amelogenin was immunohistochemically demonstrated in the enamel of developing tooth germs. Teeth of the snake are covered with a thin true enamel layer about 1-2 microns in thickness. Light and electron microscopic immunohistochemistry indicated an intense amelogenin immunoreactivity occurring in the enamel layer during the secretory stage of tooth development. Cloning and cDNA sequence of snake amelogenin was performed by RT-PCR. The amino acid sequence of the snake amelogenin cDNA--in its portion corresponding to the area from exon 5 to exon 7 of human X189 amelogenin gene--showed 45% homology with humans. Regions of both the N-terminus and C-terminus were well conserved. Furthermore, the positions of prolin in the amino acid alignment of the snake amelogenin corresponded well with those of human amelogenin. It is suggested that prolin is an essential constituent of amelogenin and therefore its positions in the molecule have been conserved after the evolutionary divergence of reptiles and mammals. This study using reptiles is the first detection of specific amelogenin immunoreactivity by high resolutional immunoelectron microscopy and the first cloning of amelogenin cDNA in a non-mammalian animal.

Delayed diagnosis of anomalous origin of the left coronary artery 16 years after mitral valve replacement.

Mitral insufficiency caused by ischemia is frequently found in anomalous origin of the left coronary artery from the pulmonary artery. We report a case of a 25-year-old woman who was diagnosed to have anomalous origin of the left coronary artery from the pulmonary artery and had successful left internal mammary artery bypass grafting 16 years after mitral valve replacement for mitral insufficiency of an unknown cause in her childhood.