Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid and high-sensitive detection of Cryptosporidium in water samples.

We describe a novel assay for simple, rapid and high-sensitive detection of Cryptosporidium oocysts in water samples using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The detection limit of the developed RT-LAMP assay was as low as 6 x 10(-3) oocysts/test tube, which theoretically enables us to detect a Cryptosporidium oocyst and perform duplicated tests even if water samples contain only one oocyst. The developed RT-LAMP assay could more sensitively detect Cryptosporidium oocysts in real water samples than the conventional assay based on microscopic observation.

Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs.

Sequence and phylogenetic analyses of three isolates of canine distemper virus (CDV) isolated from three dogs with a vaccination history were compared with the same analyses of vaccine virus isolated from a vaccine used for dogs. The three dogs showed clinical signs of a recent major type of CD in Japan, including oculonasal discharge and diarrhea, and pathological findings including non-suppurative encephalitis, pneumonia, mild gastroenteritis and lymphoid depletion. Inclusion bodies were in the stomach without inflammation and encephalitis was without clinical signs. One of the highest titers of CDV in different organs of the three dogs was commonly systemic lymphatic organs, including the spleen, lymph nodes and tonsils. New isolates of CDV joined to the clades of the Asia 1 group that is far from the vaccine group. These results surely indicate that wild strains of CDV from dogs with a vaccination history were not reversed vaccine virus, and that the dogs showed characteristics of recent CD in Japan.

Pathogenesis and phylogenetic analyses of canine distemper virus strain 007Lm, a new isolate in dogs.

The pathogenesis of a new isolate of canine distemper virus (CDV), strain 007Lm, was investigated from lymph node tissue by using Vero cells that express canine signalling lymphocyte activation molecules with a tag (Vero-DST) in dogs. Two CDV sero-negative Beagle dogs were inoculated intranasally and intraconjunctively with a virus suspension. Both infected dogs showed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, lymphopenia, fever and weight loss. Titers of CDV-IgM and CDV-IgG in the blood were measured. CDV was detected by using reverse transcriptase-PCR and was recovered in swabs from one dog from 9 days and from the other dogs from 10 days after inoculation. Molecular and phylogenetic analyses of H and P genes showed that nucleotide and amino acid sequences of these genes of strain 007Lm after isolation in Vero-DST cells are identical to those of the original virus from fresh tissue and that strain 007Lm joins to the Asia 2 group cluster of CDV strains that is distinct from other clusters. These results indicate that (1) CDV strain 007Lm isolated in Vero-DST cells is virulent, (2) nucleotide and amino acid sequences of H and P genes of strain 007Lm do not change after isolation in Vero-DST cells compared with the original virus from fresh tissue and (3) strain 007Lm isolated from a vaccinated dog belongs to a cluster far from the vaccine strains in the phylogenetic trees of H and P genes.

Experimental observation of disorder-driven hysteresis-loop criticality.

We have studied the effect of magnetic disorder on the magnetization reversal process in thin Co/CoO films. The antiferromagnetic CoO layer allows a reversible tuning of the magnetic disorder by simple temperature variation. For temperatures above a critical temperature T(c), we observe a discontinuous magnetization reversal, whereas smooth magnetization loops occur for T

Coronary arterial lesions induced by endothelin antagonists.

Endothelins are potent vasoconstrictors and pressor peptides and are important mediators of cardiac, renal and endocrine functions. Increased ET-1 levels in disease states such as congestive heart failure, pulmonary hypertension, acute myocardial infarction, and renal failure suggest the endothelin system as an attractive target for pharmacotherapy. A non-peptidic, selective, competitive endothelin receptor antagonist with an affinity for the ET(A) receptor in the subnanomolar range was administered by continuous intravenous infusion to beagle dogs, rats, and Goettingen minipigs. It caused mild arteriopathy characterised by segmental degeneration in the media of mid- to large-size coronary arteries in the heart of dog, but not rat or minipig. The lesions only occurred in the atrium and ventricle. Frequency and severity of the vascular lesions was not sex or dose related. No effects were noted in blood vessels in other organs or tissue. Plasma concentrations at steady state, and overall exposure in terms of AUC((0-24h)) were higher in minipig and rat than the dog but did not cause cardiac arteriopathy. These findings concur with those caused by other endothelin anatagonists, vasodilators and positive inotropic/vasodilating drugs such as potassium channel openers, phosphodiesterase inhibitors and peripheral vasodilators, and confirm that dogs appear to be uniquely sensitive to the development of cardiac vascular lesions.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 18). Comparative 4 and 2 weeks oral repeated dosing studies on male reproductive organs in rats treated with 5-fluorouracil.

Oral repeated toxicity studies were conducted to compare the effects of 5-fluorouracil (5-FU) administered for 4 or 2 weeks on male reproductive organs of Sprague-Dawley (SD) rats. In both studies, decrease of feces, abnormal fur and emaciation were observed. On gross autopsy examination, softening/atrophy of the testis as well as atrophy of accessory reproductive organs were noted, and absolute organ weights of male reproductive organs were almost all reduced in both studies. Histopathologically, degeneration of the seminiferous epithelium in the testis and desquamated cell debris in the epididymal ducts were apparent after both time periods. In the 2-week study, furthermore, exfoliation of the seminiferous epithelium, formation of multinucleated giant cells and vacuolation of Sertoli cells in the seminiferous tubules in the testis, and decrease of sperms in the epididymal ducts were observed. The present results indicated that a 2-week study is sufficient to detect effects on the male reproductive organs of 5-FU treatment. In our study, however, changes in associated parameters after 2-week treatment of 8-week-old rats were greater than those after 4 weeks in 6-week-old rats. Thus, for detection of 5-FU-induced male reproductive toxicity, we can evaluate more accurately by using maturated rats of the same age.

Flow cytometric analysis for sperm viability and counts in rats treated with trimethylphosphate or pyridoxine.

Six-week old SD-Slc male rats were treated for 4 weeks with compounds known to induce toxicological changes in male reproductive organs (pyridoxine in saline, 500 mg/kg/day, i.p.) or sperm (trimethylphosphate in distilled water, 100 mg/kg/day, p.o.). Each sperm sample taken from the cauda epididymis was analyzed with flow cytometry for the evaluation of sperm viability and counts. Sperm motility and morphology by microscopical observation, and histopathological examination of reproductive organs were also performed for estimating the adverse effects of each compound on spermatogenesis and sperm. While a decrease in sperm motility was noted for the trimethylphosphate group, the low motile sperm was evaluated as being viable sperm, not moribund, with flow cytometry. In the pyridoxine group, microscopical observation revealed morphological changes of sperm and a decrease of motility. The present sperm analysis with flow cytometry also suggested morphological changes reflected by dot plot as well as decrease of sperm viability and counts. These results indicated that this procedure led to profound findings in the compound-treated animals, with evaluation as viable sperm, not moribund, in a low motile sperm sample, and suggesting morphological changes in the dot plot of flow cytometry.

Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors.

SUMMARY: Mice lacking p27(Kip1) have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27(-/-) thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similar to mice with the Rb mutation, the p27(-/-) mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27(Kip1) acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFbeta, rapamycin, or contact inhibition remained intact in p27(-/-) cells, suggesting that p27(Kip1) is not required in these pathways.

Radiolucent bone cysts and the type of dialysis membrane used in patients undergoing long-term hemodialysis.

The relationship between the types of dialysis membrane used and the prevalence and severity of radiolucent bone cysts (which are a main radiological feature of dialysis amyloidosis) was studied in 30 patients on hemodialysis for more than 10 years. One of them was treated exclusively with cuprophane; the other 29 were dialyzed with cuprophane, and then treated with polyacrylonitrile AN 69. In 12 of the 30 patients, radiolucent bone cysts (at least 5 mm in diameter in the wrists and at least 10 mm in the shoulders or hips) were observed. The patients with bone cysts spent significantly more time on cuprophane dialysis and significantly less time on AN 69 dialysis than the group of patients without bone cysts. Nine of the 14 patients who had been treated with cuprophane for more than 8 years had bone cysts; whereas bone cysts were observed in only 2 of the 12 patients dialyzed for more than 8 years with AN 69. The frequency of bone cysts was significantly different for each of the two groups. There was, however, no significant difference in the total duration of dialysis between the two groups. The severity of the cystic bone lesions correlated positively with the duration of dialysis using cuprophane and negatively with the duration of dialysis using AN 69. These findings suggest that the development of osteoarticular amyloidosis may be related to the type of dialysis membrane used. Hemodialysis using AN 69 membranes may prevent, or at least postpone the development of dialysis amyloidosis.

Immunohistological localization of beta-2-microglobulin in renal tissue as an indicator of renal dysfunction.

To elucidate the relationship between localization of beta 2-microglobulin (beta 2-MG) and renal lesions or dysfunction, 119 patients with various renal diseases and various degrees of renal injuries were examined: patients with beta 2-MG deposition (group 1, n = 69), and patients without renal beta 2-MG deposition (group 2, n = 50). beta 2-MG was found mainly in the tubular epithelium and tubular casts. No significant difference in the degree of proteinuria and hematuria were found between the two groups. Group 1 had a significant decrease in glomerular filtration rate (GFR; p less than 0.01): the average values of GFR in group 1 and 2 were 61.1 +/- 35.7 and 95.4 +/- 34.5 ml/min. Group 1 had a significant decrease in the phenolsulfonphthalein excretion test (p less than 0.01) and the maximum urine specific gravity in Fishberg's concentration test (p less than 0.02). Group 1 had a significant high incidence of glomerular sclerotic lesions (p less than 0.001), arteriolar elastosis (p less than 0.01), tubulo-interstitial changes (p less than 0.001) and renal deposition of lysozyme (p less than 0.001). The present study demonstrates that the immunohistological study of renal beta 2-MG deposition is a reliable method to identify renal dysfunction and renal injuries, especially the presence of tubulo-interstitial changes, in various renal diseases.

Comparative study of IgA nephropathy with acute and insidious onset. Clinical, laboratory and pathological findings.

In order to clarify the difference of clinical and pathological features between the IgA nephropathy patients with acute and insidious onset, 427 patients were examined in this study. Seventy-eight patients with acute onset (group 1) were often associated with mucosal system infections at the abrupt onset. This group revealed macroscopic hematuria, more severe microscopic hematuria (more than 20/hpf), higher glomerular filtration rate (p less than 0.01) and lower serum levels of C3 (p less than 0.01). It had also a significantly higher incidence of exudative lesions (p less than 0.001). On the other hand, the onset of 349 patients (group 2) was noticed to be insidious without preceding infections. This group showed a more severe increase in mesangial cells (p less than 0.01) and a significantly higher incidence of adhesion, arterial sclerosis and tubulointerstitial changes. Deposition of Clq, C4 and IgM and detachment of visceral epithelium from the basement membrane were more frequently seen in group 2. Twenty-seven of 345 patients followed for at least 1 year after the biopsy were on maintenance hemodialysis: 1 patient was in group 1 and 26 were in group 2. These results clarified that there was a difference in clinical, laboratory and histopathological findings between the patients with IgA nephropathy with acute and insidious onset.

Immunohistological localization of apolipoproteins in the glomeruli in renal disease: specifically apoB and apoE.

Recently it has become clear that abnormalities of the lipid metabolism may play a large role in the progression of renal diseases. To investigate the relationship between lipids and kidney tissues, the authors employed an immunofluorescent technique to determine the presence of apolipoprotein (apo) B and E in kidney tissue, particularly the glomeruli, and analyzed the relationship between their deposition and the clinical and histological findings of a total of 49 patients with persistent proteinuria and/or hematuria (age range: 10 to 62 years). The patients were divided into 4 groups, as follows: both apoB and apoE negative cases (Group 1; 17 cases), apoB alone positive (Group 2; 7 cases), apoE alone positive (Group 3; 10 cases) and both apoB and apoE positive cases (Group 4; 15 cases). Group 2 had more severe proteinuria and a higher level of total cholesterol than Group 1. Group 3 exhibited a higher incidence of glomerular adhesion and interstitial changes than Group 1. Group 4, on the other hand, exhibited more severe mesangial hypercellularity and a higher incidence of glomerular sclerosis and interstitial scarrings than Group 1, a higher incidence of glomerular sclerosis than Group 2, more severe proteinuria, higher serum levels of total cholesterol, and lower serum levels of total protein than Groups 1 and 3 and higher level of uric acid than Group 1. These results suggest that the deposition of apoB and apoE accelerates the progression of mesangial lesions, resulting in greater proteinuria and glomerular sclerosis.