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.

[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.

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.

Central mechanism of neural activation with cold acclimation of rats using Fos immunohistochemistry.

The expression of Fos protein in the rat diencephalon, brain stem, cerebellum, and spinal cord was investigated using immunohistochemistry during chronic cold exposure, in order to clarify the neural regions involved in the thermoregulatory responses and the central mechanism of neural activation with cold acclimation. Numerous Fos-positive cells were observed in many brain regions after cold exposure and changes in the number of Fos-positive cells were analyzed quantitatively. Fos-positive regions were classified into three groups on the basis of the expression period of Fos protein. The first group was where a significant number of Fos-positive cells were seen 3 h and 24 h after cold exposure, but not observed 14 days after exposure; the regions included the lateral septal nucleus (LS), parvocellular paraventricular hypothalamic nucleus (pPVN), posterior hypothalamic area (PH), supramammillary nucleus (SuM), locus coeruleus (LC), dorsal tegmental nucleus (DTg), vestibular nucleus (Ves), and nucleus of solitary tract (Sol). The second group was where a significant number of Fos-positive cells were found 3 h, 24 h and 14 days after cold exposure; the regions included the preoptic hypothalamic area (POA), paraventricular thalamic nucleus (PV), lateral preoptic area (LPO), zona incerta (ZI), subparafascicular thalamic nucleus (SPF), lateral dorsal central grey (CGLD), lateral ventral central grey (CGLV), microcellular tegmental nucleus (MiTg), lateral lemniscus nucleus (LL), dorsal parabrachial nucleus (DPB), and the cerebellum. The third group was where Fos-positive cells were more numerous 14 days after cold exposure than they were after 3 h and 24 h of exposure; these regions included the ventromedial hypothalamic nucleus (VMH) and the spinal cord. These results demonstrate that the numbers and regions of Fos-positive cells in the rat brains changed during chronic cold exposure, and such changes may reveal the cellular adaptation of the thermogenic responsive neurons in the rat brain to cold acclimation.

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.

Temporal changes of c-fos expression in oxytocinergic magnocellular neuroendocrine cells of the rat hypothalamus with restraint stress.

The present experiments were undertaken to examine c-fos expression in magnocellular neuroendocrine cells (MNCs) of the rat hypothalamus with restraint stress using dual immunohistochemistry for c-fos and oxytocin. Restraint stress induced c-fos expression in oxytocinergic MNCs in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). Quantitative immunohistochemical analysis revealed that percentages of c-fos-positive cells to oxytocin-immunoreactive MNCs in the SON and PVN maximally increased at 2 h after restraint stress had started, and began to decline in spite of the fact that the restraint of animals were continued. Similar results were obtained from time course of c-fos expression in parvocellular neurons of the PVN. When animals were released to move freely in their home cages following the 3-h restraint, the plasma levels of oxytocin declined to reach basal levels within 30 min and c-fos immunoreactivity in the hypothalamic MNCs and parvocellular neurons disappeared faster than those of the continually restrained. These results demonstrate that restraint stress induces c-fos expression in oxytocinergic MNCs in the SON and PVN, and that time course of c-fos expression is transient even in the continuation of restraint stress.

Stability of a metabolizable ester bond in radioimmunoconjugates.

Ester bonds have been used as metabolizable linkages to reduce radioactivity levels in non-target tissues following the administration of antibodies labeled with metallic radionuclides. In this radiochemical design of antibodies, while the ester bonds should be cleaved rapidly in non-target tissues, high stability of the ester bonds in plasma is also required to preserve target radioactivity levels. To assess the structural requirements to stabilize the ester bond, a new benzyl-EDTA-derived bifunctional chelating agent with an ester bond, (1-[4-[4-(2- maleimidoethoxy)succinamido]benzyl]ethylenediamine-N,N,N',N' -tetraacetic acid; MESS-Bz-EDTA), was developed. MESS-Bz-EDTA was coupled with a thiolated monoclonal antibody (OST7, IgG1) prepared by reducing its disulfide bonds to introduce the ester bond close and proximal to the antibody molecule. For comparison, 1-[4-(5- maleimidopentyl)aminobenzyl]ethylenediamine-N,N,N',N'-tetraacetic acid (EMCS-Bz-EDTA) and meleimidoethyl 3-[131I]iodohippurate (MIH) was coupled to OST7 under the same conjunction chemistry. When incubated in 50% murine plasma or a buffered-solution of neutral pH, OST7-MESS-Bz-EDTA-111In rapidly released the radioactivity, and more than 95% of the initial radioactivity was liberated after a 24 h incubation in both solutions, due to a cleavage of the ester bond. On the other hand, only about 20% of the radioactivity was released from OST7-MIH-131I in both solutions during the same incubation period. In mice biodistribution studies, while a slightly faster radioactivity clearance from the blood with less radioactivity levels in the liver and kidneys was observed with OST7-MIH-131I than with OST7-EMCS-Bz-EDTA-111In, OST7-MESS-Bz-EDTA-111In indicated radioactivity clearance from the blood much faster than and almost comparable to that of OST7-MIH-131I and succinamidobenzyl-EDTA-111In, respectively. These findings as well as previous findings on radiolabeled antibodies with ester bonds suggested that while an introduction of an ester bond close to an antibody molecule stabilized the ester bond against esterase access, chemical structures of the linkages and radiolabels attached to the ester bonds play a significant role in the chemical stability of the ester bond. This may explain the different stability of the ester bonds in radioimmunoconjugates so far reported.

The structure and development of the collar enameloid in two teleost fishes, Halichoeres poecilopterus and Pagrus major.

Histologically the outer layer of the collar enameloid obviously differs from the inner layer, and it has a degree of mineralization nearly as high as the cap enameloid which has the highest. In the stage of matrix formation, the organic matrix of the collar enameloid contains a number of collagen fibers, and odontoblasts display features suggesting that these cells actively synthesized and secreted collagen. A number of cell processes, matrix vesicles and some cell debris which were probably derived from the odontoblasts were observed in the organic matrix of the collar enameloid. We consider that the majority of the organic matrix in collar enameloid originates from the odontoblasts. In the stage of maturation, collagen fibers were not observed in the outer layer of the collar enameloid in demineralized specimens. In the IDE cells during this stage, the complex infoldings of cell membranes developed in the distal portion, and several lysosomal granules and irregular-shaped granules containing many tubular structures, were observed in the distal cytoplasm. In the ODE cells, abundant labyrinthine canals appeared in the cytoplasm, and capillary vessels were found close to the outer surface of the ODE cells. We assume that the higher mineralized outer layer of the collar enameloid is made possible by the absorptive and transport functions of the epithelial cells during the stage of maturation. It is considered that the collar enameloid in this study was initially produced by the odontoblasts and then reconstructed by the epithelial cells, so that the collar enameloid differs from true enamel.

Microstructural features of dipnoan tooth enamel.

Tooth plates of extant lungfishes were examined by contact microradiography, light microscopy and TEM, to elucidate the nature and origin of the hypermineralized surface layer. TEM indicated that the matrix of this layer consisted of organic filamentous material, and no collagen fibres were seen in the matrix. The matrix appeared to be secreted by oral epithelial cells of the inner fold surrounding the surface of the tooth plate, and deposited in the area between the cell membrane and basal lamina-like structure of these epithelial cells. Therefore, it is suggested that the hypermineralized surface layer of lungfish plates is enamel of ectodermal origin.

A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability.

A highly water soluble disulfonated tetrazolium salt, 4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt, was synthesized. The compound is reduced by NADH in good yields at neutral pHs in the presence of 1-methoxy PMS to produce the corresponding formazan dye that absorbs at 460 nm. The formazan is soluble to water at concentrations higher than 0.1 M. The tetrazolium salt thus proved to be useful as a sensitive chromogenic indicator for NADH. It is also applicable to cell proliferation assays as a cell viability indicator.

[A case report: reopacified thrombosed dissection of Stanford type A].

A 60-year-old male, who had been hospitalized with a Stanford type B acute aortic dissection 3 years ago, was admitted with severe disquiet caused by discomfort of the chest and the neck. Enhanced CT scans demonstrated the ascending aorta compressed by non-opacified false lumen, so-called "thrombosed dissection" or "closing aortic dissection", and normal descending aorta. Aortography showed no intimal tear in the ascending aorta and aortic arch. "Hibernation therapy" with pentobarbital was performed to be sedated over during 13 days. 29 days later follow-up CT scans showed reopacified false lumen and enlargement of the ascending aorta, although he had no complaints during the medical therapy. Large intimal tear just under the brachiocephalic artery and giant false lumen without reentry occupying the ascending aorta was found by the repeat aortography. Graft replacement of the ascending aorta was successfully performed. This case suggested that thrombosed dissection might have the risk of recanalization of clotted false lumen asymptomatically, and it would be important to take care of radiographic changes during the medical therapy even if the patient had no symptoms.

[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.

[Surgical relief of airway obstruction from a double aortic arch associated with corrected transposition of the great arteries, pulmonary atresia and bilateral patent ductus arteriosus in a neonate].

A rare 20-day-old male with double aortic arch, corrected transposition of the great arteries (cTGA), pulmonary atresia and bilateral patent ductus arteriosus (PDA) was transported to our institute because of severe respiratory dysfunction and cyanosis. The patient had been already intubated and ventilated on respirator. A echocardiography and cine-angiography demonstrated that the both sides aortic arch had almost identical sizes, originating common carotid arteries and subclavian arteries and PDAs respectively, and the descending aorta located on the left side of the mid-line. At the first surgery, the distal of the right aortic arch was divided just proximal to the descending aorta after complete tissue dissection around the arch. The divided right sided aortic arch was mobilized from posterior to anterior aspect of the bronchus. Then the right subclavian artery was divided and an original Blalock-Taussig shunt was employed. The right sided PDA was ligated. After the first surgery, respiratory dysfunction lasted for weeks mainly because of the PGE1 dependent left sided PDA. At the second surgery, left sided modified Blalock-Taussig shunt was constructed and the left sided PDA was divided. These procedures resulted in stable respiratory status and oxygen saturation. The patient was extubated three days later and now in satisfactory clinical condition.

[Effects of 5-fluorouracil in shrinking residual gastric cancer: a case report].

The patient was a 79-year-old man who underwent extensive gastrectomy and Billroth-II reconstruction for gastric ulcer in 1974. He complained of heartburn in November, 1992, and endoscopy for the residual stomach revealed IIb + IIa-like lesion mainly in the posterior wall of the anastomosed site, which was diagnosed as well differentiated adenocarcinoma. As the patient refused to be operated, 150 mg/day of 5-FU was administered. For 4 months before he decided to undergo operation, 12.9 g of 5-FU was administered in total. After rehospitalization, endoscopy of the residual stomach showed shrinkage of the lesion, and biopsy revealed no malignant findings. The pathological examination after total resection of the residual stomach showed atypism, but no evidence to support malignancy. He has continued to be well without sign of recurrence for 1 year since the operation. The administration of 5-FU is widely used as preoperative adjuvant chemotherapy. This case it is rare for 5-FU to cause the disappearance or falling off of malignant cells.

[Postsplenectomy infection and immunologic aspects of splenectomy].

Of 619 trauma patients undergoing splenectomy, 503 lived more than 10 days. In this group, 114 patients (22.7%) developed systematic sepsis and 36 patients (7.2%) succumbed from sepsis. Septic morbidity and mortality rates in splenectomized patients were significantly greater than those in 2180 consecutive trauma patients treated in 1978-1979. Septic mortality rate in septic patients was significantly greater than that in 2180 consecutive trauma patients. Septic mortality rate in the patients whose trauma were seen only in the spleen or associated with only one organ injury was also very high. Long-term follow-up information was obtained in 242 splenectomized patients with a mean patient follow-up interval of 4.4 years. Severe bacterial infections have occurred in six patients. Thus far, there have been no deaths from overwhelming sepsis. Of interest, 10% of the patients complained of more severe viral infection following splenectomy. On the other hand, the incidence of postoperative infection in the patients who underwent total gastrectomy with splenectomy was not different from that of the patients who underwent total gastrectomy without splenectomy. However, the incidence of the postoperative infection in the splenectomized patients together with total gastrectomy was greater than that of the the splenectomized patients in trauma.