Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum.

UNLABELLED: This study reports the development of a loop-mediated isothermal amplification (LAMP) reaction for the detection of Pythium myriotylum. The primer set targeting the ITS sequence of P. myriotylum worked most efficiently at 60°C and allowed the detection of P. myriotylum DNA within 30 min by fluorescence monitoring using a real-time PCR instrument. The peak denaturing temperature of amplified DNA was about 87·0°C. In specificity tests using eight Pythium myriotylum strains, 59 strains from 39 species of Pythium, 11 Phytophthora strains and eight other soil-borne pathogens, LAMP gave no cross-reactions. The detection limit was 100 fg of genomic DNA, which was as sensitive as PCR. LAMP could detect P. myriotylum in hydroponic solution samples, and the results coincided with those of the conventional plating method in almost all cases. The LAMP method established in this study is a simple and sensitive tool for the detection of P. myriotylum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the first LAMP assay for the detection of Pythium myriotylum. The primer set designed from ITS region of P. myriotylum can detect the pathogen in field sample with a fast and convenient method. Analysis of the annealing curve of the LAMP reaction products increases the reliability of the LAMP diagnosis. This study shows that the diagnostic method using the LAMP assay is useful for monitoring P. myriotylum in the field.

The surgical treatment of pyogenic spondylitis using posterior instrumentation without anterior debridement.

There have been few reports regarding the efficacy of posterior instrumentation alone as surgical treatment for patients with pyogenic spondylitis, thus avoiding the morbidity of anterior surgery. We report the clinical outcomes of six patients with pyogenic spondylitis treated effectively with a single-stage posterior fusion without anterior debridement at a mean follow-up of 2.8 years (2 to 5). Haematological data, including white cell count and level of C-reactive protein, returned to normal in all patients at a mean of 8.2 weeks (7 to 9) after the posterior fusion. Rigid bony fusion between the infected vertebrae was observed in five patients at a mean of 6.3 months (4.5 to 8) post-operatively, with the remaining patient having partial union. Severe back pain was immediately reduced following surgery and the activities of daily living showed a marked improvement. Methicillin-resistant Staphylococcus aureus was detected as the causative organism in four patients. Single-stage posterior fusion may be effective in patients with pyogenic spondylitis who have relatively minor bony destruction.

Abundance of calpain and aggrecan-cleavage products of calpain in degenerated human intervertebral discs.

OBJECTIVE: To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs). DESIGN: Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and µ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA. RESULTS: Discs at early stages of degeneration expressed low levels of m- and µ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, µ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous. CONCLUSIONS: The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.

Sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of peach latent mosaic viroid (PLMVd) was developed. Four primer sets (OLD, OLD1, NEW, and Fukuta's) were designed originally. Based on initial experiments the set OLD1 was selected for further evaluation. Simple and accelerated RT-LAMP was preformed using degenerate and no degenerate forward-loop (F-loop) and backward-loop (B-Loop) primers. Degenerate primers were selected, and after determination of their best concentration (0.8microM), the reaction was preformed at different temperatures (60-67.5 degrees C) using three different betaine concentrations (0.8M, 0.4M, and 0.2M). Optimal conditions were found to be 62.5 degrees C and 0.8M betaine. Under these conditions, using tRNA as template, PLMVd was detected within only 32min, compared to 180min of RT-PCR, using the Real Time Turbimeter (LA200, Teramecs) which measures the turbidity caused by the production of insoluble magnesium pyrophosphate. In addition, RT-LAMP was more sensitive than RT-PCR. PLMVd was detected in peach, plum, apricot, pear, wild pear and quince samples.

Detection of Japanese yam mosaic virus by RT-LAMP.

Arapid and simple procedure is described to detect the genomic RNA molecule of Japanese yam mosaic potyvirus (JYMV). This method, named RT-LAMP, allows direct detection of RNA from infected plants without careful RNA extraction, rapid thermal cycling and gel electrophoresis. RT-LAMP was successfully applied to leaves, propagules and roots of Japanese yam infected with JYMV. One of the characteristics of the RT-LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophospate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of the amplification of JYMV genomic RNA without gel electrophoresis.

Retrovirally transduced bone marrow stromal cells isolated from a mouse model of human osteogenesis imperfecta (oim) persist in bone and retain the ability to form cartilage and bone after extended passaging.

Bone marrow stromal cells isolated from a model of osteogenesis imperfecta (oim) mice, were transduced with a retrovirus (BAG) carrying the LacZ and neor genes after passage 21. The transduced cells retained the ability to express alkaline phosphatase activity in vitro when treated with recombinant human bone morphogenetic protein two (rhBMP-2), formed cartilage in vitro in aggregate cultures and formed bone in ceramic cubes after 6 weeks of implantation in nude mice. X-gal staining of ceramic cubes seeded with the transduced cells demonstrated the presence of LacZ-positive cells on the edges of bone and also in the lacunae of the newly formed bone 6 weeks after implantation. After infusion into femurs of oim mice, the transduced cells were detected in the marrow cavity and on the edges of the trabecular bone of the injected and contralateral femurs by X-gal staining and PCR analysis at 4, 10, 20, 30 and 40 days after injection. The LacZ gene was also detected in the lung and liver of the recipient mice at 4 and 10 days after injection but not at later time-periods. The present findings suggest that long-term cultured bone marrow stromal cells from osteogenesis imperfecta (OI) animals have the potential to traffic through the circulatory system, home to bone, form bone and continue to express exogenous genes. These findings open the possibility of using these cells as vehicles to deliver normal genes to bone as an alternative approach for the treatment of some forms of OI and certain other bone acquired and genetic diseases.

Identification of types II, IX and X collagens at the insertion site of the bovine achilles tendon.

Achilles tendinous collagen fibrils insert into the calcaneus by first passing through a zone that is defined histologically as fibrocartilaginous. This zone consists of four regions: tendon proper, non-mineralized and mineralized fibrocartilage and bone. The function of this zone has not yet been clearly defined. To gain more insight into the role of this fibrocartilaginous zone, collagens present in the zone of the Achilles tendon-calcaneus interface were isolated and characterized. Types II, IX and X collagens were identified in the pepsin digests of the tissue harvested from the bovine Achilles tendon-calcaneus interface. Western blotting using specific antisera to types II, IX and X collagens confirmed the identity of these collagens. Immunofluorescence localization placed type X collagen predominantly in the mineralized zone of the tendon-calcaneus junction, while type IX collagen was distributed throughout the the insertion site. The presence of the cartilage-specific collagens at the Achilles tendon-calcaneus-interface suggests that this zone is cartilaginous in nature. The presence of type X collagen at this junction is not clear, but our present findings go along with the previous report which showed that type X collagen is present in the mineralized zone of the medial collateral ligament femoral insertion site. These data suggest that type X collagen may be a resident of mineralized fibrocartilaginous zones of tendon or ligament-bone junctions and may participate in anchoring ligament or tendon to bone.

[A case of lupus myocarditis and nephritis with transient foramen jugular syndrome].

A 46-year-old man was admitted to our clinic because of acute heart failure. Six years before admission he was pointed out cardiomegary and hematuria. One year later, he was diagnosed as having jugular foramen syndrome. On admission, he had a fever and dyspnea. Pansystolic blowing murmur was audible at the apex. The chest ratio on his chest X-ray was 52.5%. An electrocardiogram showed left ventricular hypertrophy. An echocardiogram showed marked dilatation and severe dysfunction of left ventricle. Radionuclide scanning with technetium 99 m pyrophosphate identified inflammatory change in the apex. Myocardial biopsy showed fibrotic degeneration and IgG deposits in myocardium. Blood examination showed anemia, lymphopenia. positive anti-nuclear antibody (1000 times, shaggy pattern), positive anti ds-DNA antibody and hypocomplementemia. Furthermore, proteinuria was pointed out. Renal biopsy showed focal segmental glomerulonephritis with active necrotizing lesion (type III nephritis). Lupus myocarditis and nephritis was diagnosed. After prednisolone (80 mg/day) was administered. left ventricular function and hypocomplementemia improved. The ACE inhibitor was also used for proteinuria. In spite of a little amount of blood transfusion, he showed hepatic hemosiderosis. We suspect that the cause of hemosiderosis was related chronic inflammation of active lupus. It was treated with Erythropoietin.

A case of pleomorphic adenoma of the epiglottis. Bilateral vocal-cord paralysis after YAG laser surgery.

Pleomorphic adenoma of the larynx is a rare disorder, and until recently has been treated mainly by approaches involving pharyngotomy. We encountered a case of pleomorphic adenoma originating from the laryngeal surface of the epiglottis and removed it using a YAG laser through a suspension laryngoscope. This case was complicated by delayed-onset bilateral vocal-cord paralysis, the causes of which are also discussed.

Inner ear disorders due to pressure change.

We reviewed the records of 136 patients who had inner ear disorders including hearing loss and vertigo caused by pressure change. We divided them into three groups, according to the aetiology: group A, change in atmospheric pressure (diving, airplane travel, etc.); group B, rapid change in ear pressure in normal atmosphere (nose blowing, heavy lifting, etc.); and group C, blast injury. A flat initial audiogram was the most common type in groups A and B. In group C, high-tone hearing loss was the most common type of audiogram. These results correspond to findings previously reported in animal experiments. Exploratory tympanotomy was performed more than 12 days after the pressure change in 16 patients. Although the vertigo disappeared after surgery, hearing did not improve.

Application of a new anticoagulant (Nafamostat Mesilate) to control hemorrhagic complications during extracorporeal membrane oxygenation--a preliminary report.

Bleeding related to systemic heparinization has been considered one of the major complications associated with extracorporeal membrane oxygenation (ECMO). Development of the heparin-bonded system will be essential in reducing hemorrhagic complications, but has not yet been clinically proven. The authors chose an alternative approach of making a difference in the activated clotting time (ACT) values between the patient and the ECMO circuit, and decreased only the patient's ACT value, while keeping the value of the ECMO circuit at an ideal level. For this purpose, we have used a very short-life anticoagulant, Nafamostat Mesilate (FUT), while decreasing the dose of heparin. FUT is a synthetic protease inhibitor that has been found to inhibit various kinds of enzyme activities for coagulation. Twelve newborns who had some hemorrhagic complications at various sites before or during ECMO, were selected to receive FUT. The heparin dose was decreased after FUT administration into the drainage route. FUT and heparin doses were regulated to maintain the ACT value at the reinfusion route at 190 to 220 seconds. ACT values at the drainage and the reinfusion routes were simultaneously measured. The average time on FUT was 100.3 +/- 86.3 (SD) hours. The average dose of FUT was 0.48 +/- 0.22 mg/kg/h, and that of heparin was 21.0 +/- 7.5 U/kg/h. The average ACT value at the reinfusion route was 205.7 +/- 14.0 seconds compared with that at the drainage route of 178.5 +/- 11.8. The difference was statistically significant (P < .001). The average difference in ACT values between both routes was 27.1 +/- 7.9 seconds. The bleeding was well controlled by FUT administration in 8 of 12 cases. This report may represent the first clinical use of FUT in neonatal ECMO, and serve as a preliminary study.

Morphological studies on middle ear barotrauma in guinea pigs.

Experimental middle ear barotrauma was studied morphologically. White guinea pigs were placed in an experimental hyperbaric chamber, and middle ear barotrauma was created by increasing the pressure in the hyperbaric chamber from 1 atmosphere absolute (ATA) to 2 ATA using pure oxygen, maintaining the pressure at 2 ATA for 10 minutes, then again reducing the pressure to 1 ATA. Selected experimental animals were decapitated immediately after, one day after, one week after, or weeks after pressure loading, and their middle ears were examined by a light microscope (LM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM). Hemorrhaging in the tympanic cavity immediately after pressure exposure was apparent even macroscopically. LM also revealed evidence of submucous hemorrhage. Submucous edema was seen in the "one week after" cases. SEM showed a minor loss of cilia in some ciliated cells just after the experiment. In nonciliated cells, the terminal web was somewhat indistinct in the "one week after" cases. TEM also indicated a minor loss of cilia in some ciliated cells in "one day after" cases as well as apparent vacuoles within the cells. These findings suggest that although trauma during compression is more marked than during decompression, recovery from this damage progressed with time.

Autoimmune response in chronic ongoing myocarditis demonstrated by heterotopic cardiac transplantation in mice.

BACKGROUND: Autoimmune mechanisms have been implicated in the pathogenesis of chronic ongoing myocarditis. To investigate this relation, we used an A/J mouse model inoculated with coxsackievirus B3 and determined whether myocarditis would be transferred to normal hearts that were heterotopically transplanted. METHODS AND RESULTS: Inbred 3-week-old A/J mice were inoculated intraperitoneally with coxsackievirus B3 (Nancy strain; 2 x 10(4) plaque-forming units) and housed for > 60 days. The presence of the viral genome in the myocardium was determined by the polymerase chain reaction with primers specific for the 5' end of the coxsackievirus B3 genome performed at 40, 50, or 60 days after inoculation. Normal A/J mouse hearts were transplanted into the same strain of mice without myocarditis (group A) and into mice with chronic ongoing myocarditis (group B). The hearts were evaluated histologically 2 weeks after transplantation. Conventional histological examination of infiltrated T cells and macrophages was performed, and the expression of intercellular adhesion molecule-1, major histocompatibility complex (MHC) class I antigen, and MHC class II antigen was evaluated by immunoenzymatic staining. The concentrations of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF-alpha) in the grafts were measured with an ELISA. The viral RNA genomes were not detected in the mice with chronic ongoing myocarditis, but their transplanted hearts did show myocarditis. In the hearts with induced myocarditis, infiltrated mononuclear cells consisted of CD4+ T cells, CD8+ T cells (CD4+ cell number > CD8+ cell number), and macrophages. Intercellular adhesion molecule-1, MHC class I antigen, and MHC class II antigen were expressed in the vascular endothelial cells and myocardial cells in and around the infiltrated lesions. The concentrations of IL-1 alpha and TNF-alpha in group B were significantly higher than those in group A (group A versus group B: IL-1 alpha, 125 +/- 35 versus 180 +/- 34 pg/mL; TNF-alpha, 45 +/- 15 versus 96 +/- 40 pg/mL; P < .05). CONCLUSIONS: Results suggest that an autoimmune response may play a key role in the progression of chronic ongoing myocarditis.

A pathogenic mechanism of chronic ongoing myocarditis.

To clarify the pathogenetic mechanism of chronic ongoing myocarditis, we produced Coxsackievirus B3-induced myocarditis in A/J mice and immunopathologically examined the microcirculation in the chronic phase of myocarditis. Forty-two 3-week-old A/J mice were inoculated intraperitoneally with Coxsackievirus B3 (Nancy strain) 2 x 10(4) PFU (plaque-forming units) and sacrificed 7, 14, 21, 50, 90, or 120 days later. To evaluate myocardial microcirculation, 18 of the hearts were perfused from the thoracic aorta with warm 2% gelatin/carbon solution. The remaining hearts were quickly frozen for immunologic analysis with an enzyme immunostaining assay using monoclonal antibodies against CD4, CD8, macrophages, intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex class I or II. The presence of viral RNA genome in the myocardium at 40, 50, or 60 days after inoculation was evaluated using the polymerase chain reaction. The lesions in chronic ongoing myocarditis consisted of myocardial damage, myocardial calcification, interstitial fibrosis, and infiltration of mononuclear cells. These infiltrated lymphocytes were predominantly CD4+ T cells. Furthermore, microvascular abnormalities, including dilatation, tortuosity, constriction, and abrupt termination, were observed around the lesions. There was marked infiltration by mononuclear cells around the microvessels. ICAM-1 was strongly expressed in the endothelial cells of the vessels. Coxsackie B3 viral genome was not detected in the myocardium of mice with chronic ongoing myocarditis in each stage examined. These results suggest that an autoimmune mechanism is involved in the persistent inflammation seen in chronic ongoing myocarditis.

[Scanning electron microscopic study of inner ear barotrauma: in the guinea pig under hypobaric pressure].

In order to investigate the mechanism of inner ear barotrauma, guinea pigs, with bilateral eustachian tube occlusion, were subjected to decompression and compression between 760 and 460 mmHg in a hypobaric pressure chamber. We divided the guinea pigs into two groups, A and B. Group A showed normal eustachian tubes, and group B showed bilaterally occluded eustachian tubes. Group B animals were divided into three types according to the rates of compression and decompression. After pressure loading, morphological changes in the hair cells of the organ of Corti were studied by means of scanning electron microscopy. There was no damage to hair cells in the setting of normal eustachian tube function, as in group A. On the other hand, mild to severe hair cell damage was observed with rapid decompression in group B. This observation suggests that relative positive pressure in the middle ear cavity is an important factor in inner ear barotrauma. The mechanism of hair cell damage due to inner ear barotrauma is presumed to be distortion of the organ of Corti caused by a difference in pressure between perilymph and endolymph resulting in injury to the stereocilica.

[Vestibular changes due to barotrauma].

Morphological vestibular changes caused by barotrauma were studied in guinea pigs. Animals were exposed to rapid decompression from 2 absolute atmospheric pressures (ATA) to 1 ATA, which causes inner ear barotrauma in the guinea pig. During decompression, spontaneous nystagmus was recorded, which consisted of irritative symptoms initially, followed by paralytic nystagmus. After pressure loading and observation to confirm the absence of Preyer's reflex with vertigo, the animals were tested for caloric nystagmus using ice water and then sacrificed at varying intervals. Then, morphological changes in vestibular organs and the organ of Corti were studied. Half of the experimental animals showed canal paresis on caloric testing. Damage to the organ of Corti was severe while that to vestibular organs was very slight. Damage to the sensory cells of the vestibular organs was not clear on light microscopy, despite a partial collapse of labyrinthine membranes. Under scanning electron microscopy, local damage was observed in a portion of the crista ampullaris of the semicircular canals. In this area, incomplete or complete disappearance of kinocilia and stereocilia, similar to that seen after rotatostimulation, was observed. However, no damage to sensory hairs was seen in the utricles and saccules. The observed vestibular organ damage, resulting from inner ear barotrauma, suggested effects on endolymphatic flow.

Genetic analysis of dilated cardiomyopathy--HLA and immunoglobulin genes may confer susceptibility.

To identify genetic factors in the immune system which control the susceptibility to dilated cardiomyopathy (DCM), HLA class II DNA typing was performed in 61 Japanese patients, using PCR/SSO probe analyses. The frequencies of HLA-DQB1*0503 (15% vs 5%; RR = 3.06, chi 2 = 7.19) and DQB1*0604 (21% vs 10%; RR = 2.41, chi 2 = 6.20) were significantly increased and that of HLA-DQB1*0502 (RR = 1.74) was slightly increased in the DCM patients. The frequency of DQB1*0303 (16% vs 31%; RR = 0.44, chi 2 = 5.16) was significantly decreased in the patients. The increased HLA-DQB1 alleles have a histidine residue in common at the 30th codon for the HLA-DQ beta chain. Among the genetic markers studied by Southern blot analyses, IGLV (immunoglobulin lambda light chain, pV3.3) showed a strong association with DCM, i.e. A2/A2 genotype was found in 37.7% of patients whereas it was observed in only 18.9% of the control subjects (RR = 2.6, chi 2 = 7.77). The frequency of this genotype was higher in patients under age 45 years at the time of diagnosis (45.5%, RR = 3.6, chi 2 = 10.02). These results suggest that HLA and immunoglobulin genes are closely linked to susceptibility to DCM.