A Japanese Multicenter Study on PET and Other Biomarkers for Subjects with Potential Preclinical and Prodromal Alzheimer's Disease.

BACKGROUND: PET (positron emission tomography) and CSF (cerebrospinal fluid) provide the "ATN" (Amyloid, Tau, Neurodegeneration) classification and play an essential role in early and differential diagnosis of Alzheimer's disease (AD). OBJECTIVE: Biomarkers were evaluated in a Japanese multicenter study on cognitively unimpaired subjects (CU) and early (E) and late (L) mild cognitive impairment (MCI) patients. MEASUREMENTS: A total of 38 (26 CU, 7 EMCI, 5 LMCI) subjects with the age of 65-84 were enrolled. Amyloid-PET and FDG-PET as well as structural MRI were acquired on all of them, with an additional tau-PET with 18F-flortaucipir on 15 and CSF measurement of Aß1-42, P-tau, and T-tau on 18 subjects. Positivity of amyloid and tau was determined based on the positive result of either PET or CSF. RESULTS: The amyloid positivity was 13/38, with discordance between PET and CSF in 6/18. Cortical tau deposition quantified with PET was significantly correlated with CSF P-tau, in spite of discordance in the binary positivity between visual PET interpretation and CSF P-tau in 5/8 (PET-/CSF+). Tau was positive in 7/9 amyloid positive and 8/16 amyloid negative subjects who underwent tau measurement, respectively. Overall, a large number of subjects presented quantitative measures and/or visual read that are close to the borderline of binary positivity, which caused, at least partly, the discordance between PET and CSF in amyloid and/or tau. Nine subjects presented either tau or FDG-PET positive while amyloid was negative, suggesting the possibility of non-AD disorders. CONCLUSION: Positivity rate of amyloid and tau, together with their relationship, was consistent with previous reports. Multicenter study on subjects with very mild or no cognitive impairment may need refining the positivity criteria and cutoff level as well as strict quality control of the measurements.

Identification of independent APP locus duplication in Japanese patients with early-onset Alzheimer disease.

BACKGROUND: The occurrence of duplications of the amyloid precursor protein gene (APP) has been described in European families with early-onset familial Alzheimer disease (EO-FAD) and cerebral amyloid angiopathy. However, the contribution of APP duplication to the development of AD in other ethnic populations remains undetermined. METHODS: The occurrence of APP duplication in probands from 25 families with FAD and 11 sporadic EO-AD cases in the Japanese population was examined by quantitative PCR and microarray-based comparative genomic hybridisation analyses. APP expression level was determined by real-time quantitative reverse-transcription (RT) PCR analysis using mRNA extracted from the peripheral blood of the patients. RESULTS: We identified APP locus duplications in two unrelated EO-FAD families. The duplicated genomic regions in two patients of these families differed from each other. No APP duplication was found in the late-onset FAD families or sporadic EO-AD patients. The patients with APP duplication developed insidious memory disturbance in their fifties without intracerebral haemorrhage and epilepsy. Quantitative RT-PCR analysis showed the increased APP mRNA expression levels in these patients compared with those in age- and sex-matched controls. CONCLUSIONS: Our results suggest that APP duplication should be considered in patients with EO-FAD in various ethnic groups, and that increased APP mRNA expression level owing to APP duplication contributes to AD development.

Chronic myositis with cardiomyopathy and respiratory failure associated with mild form of organ-specific autoimmune diseases.

We report the four patients with chronic myositis characterized by a very slow progression with cardiomyopathy and frequently with marked respiratory muscle weakness associated with other organ-specific autoimmune diseases such as primary biliary cirrhosis. The histopathology of the muscle showed many degenerative and regenerative fibers, but inflammatory-cell infiltration were minimal. The patients showed favorable response to high-dose corticosteroid treatment. Because of these clinical features, these patients are sometimes misdiagnosed as muscular dystrophy and not treated properly. It is important to distinguish this type of treatable myositis.

Cloning and characterization of genes encoding an enzyme which oxidizes dimethyl sulfide in Acinetobacter sp. strain 20B.

Acinetobacter sp. strain 20B was isolated based on the ability to utilize dimethyl sulfide as the sole sulfur source. Since strain 20B oxidized indole as well as dimethyl sulfide, indigo production by recombinant Escherichia coli clones carrying Acinetobacter DNA was used as a selection for cloning genes encoding dimethyl sulfide oxidation genes. The gene encoding an indole-oxidizing enzyme was also found to oxidize dimethyl sulfide. The dimethyl sulfide-oxidizing enzyme genes consisted of six open reading flames designated dsoABCDEF. The deduced amino acid sequences of dsoABCDEF were homologous with those of the multicomponent phenol hydroxylases. DsoABCDEF oxidized dimethyl sulfide to dimethyl sulfoxide, and dimethyl sulfoxide to dimethyl sulfone.

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards.

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.

[Detection of rubella virus antibody by enzyme linked immunosorbent assay with parallel line method].

An attempt was made to measure the antibody to rubella virus in human sera by ELISA by calculating the relative potency by the method of the parallel line assay. As a standard serum, adult serum 8 months after rubella infection was used. Sample sera were collected from 42 infants before MMR vaccination and 8-10 weeks after the vaccination and 48 adults three years or more after rubella vaccination. In addition, three infant sera with rubella 6-19 days after onset of rash and three gamma immunoglobulin products were used. The dose response curve of the standard serum and that of the sample serum showed the lineality and the regression line of the sample serum was parallel to that of the standard serum. And the relative potency of sample sera was successfully calculated. The good correlation was observed between the rubella antibody titer by hemagglutination inhibition test and that measured by ELISA. The coefficients of variance of relative potency obtained from four repeated measurements for two weeks showed from 15% to 25% in four sera of HI high titer. The reproducibility of parallel line assay was also satisfactory.

Use of subjective estimation in motor skill tests of young children: judgment based on observation of behavior in daily life.

This study assessed what motor skill tests were appropriate by observing 636 young children's behavior in daily life (low-aged classes, M = 3.7 yr.; middle-aged classes, M = 4.7 yr.; high-aged classes, M = 5.7 yr.). A homeroom teacher and an assistant teacher estimated motor achievement as pass-or-fail, and then judged pass-or-fail based on practical testing. Estimate-re-estimate agreement, interrater agreement, and agreement between estimated values and measured values were examined for 27 items, e.g., skipping, bouncing a ball, and turning on one leg. Estimate-re-estimate agreement was high on the whole. Interrater agreement ranged from 34% to 100% for 3-yr.-olds, 21% to 100% for 4-yr.-olds, and 89% to 100% for 5-yr.-olds. Agreement between estimated values and measured values greater than 80% was found in most items for 5-yr.olds (14 items). After examining the above-mentioned agreements, 26 items were selected as possible tests to judge motor development, using a pass-or-fail, from the observation of young children's behavior in daily life.

Selection of effective tests of motor ability in preschool children based on pass-or-fail criteria: examination of reliability, objectivity, and rate of passing.

The purpose of this study was to select effective tests of motor ability based on pass-or-fail criteria for use with preschool children. 37 items selected by examining theoretical validity and the results of preliminary tests were administered to preschool children (3 yr.: M = 3.7 yr., SD = 0.28; 4 yr.: M = 4.7 yr., SD = 0.28; 5 yr.: M = 5.7 yr., SD = 0.28). A skilled tester and each child's homeroom teacher rated whether the child's performance passed certain criteria or not. With agreement on two trials as an index of test-retest reliability, the mean agreement among the three grades ranged from 69% to 99% for Locomotion, 59% to 95% for Manipulation; and 66% to 100% for Stability. Disagreement on two trials may reflect instability in movement, practice effects, and so on. With agreement between two testers as an index of objectivity for 37 items, 33 showed interrater agreement of 80% or more for all three grades. No significant increase in pass rate with age was found on 10 items. In examining the three conditions mentioned above 27 items were selected as tests of motor ability: 14 items for Locomotion, 7 items for Manipulation, and 6 items for Stability.

[Development of ADL index for partially dependent older adults].

The purpose of this study was to develop an index of activities of daily living (ADL) for partially dependent older adults (PD). The index is intended to assess the capability of PD in performing basic daily tasks. A questionnaire of 22 items representing 7 ADL domains was administered by institutional therapists (nurse, OT, PT, social worker) on 466 PD 60 years or more of age, excepting completely dependent people. As a result of examining achievement rates and sex/age differences, inter- and intra-rater reliability (perfect agreement rates), and scalability, 17 reliable items were selected. The validity, reliability and unidimensionality of the ADL index using the 17 items were verified. Age and ADL score calculated based on Hosokawa's extended ADL scale were used as the external standard of validity. Furthermore, the relationships between ADL score and self-assessment of health status and physical fitness level were examined. The main findings of this study can be summarized as follows: 1) The reliability of the ADL index using 17 items was high (r = 0.996 in inter-reliability, and r = 0.940 in intra-reliability). Cronbachs' alpha coefficient of the ADL index was also high (0.926). 2) Based on scalogram analysis, the Guttman's CR, an index of unidimensionality, was very high (CR = 0.939, CS = 0.691). 3) The validity examination showed that ADL score tended to decrease significantly with age, and had a high correlation with Hosokawa's extended ADL scale (r = 0.939). 4) The ADL score tended to be higher in people with a higher self-assessment of health status and physical fitness level.

[Characteristics of each activity domain comprising ADL for partially dependent older adults].

The purpose of this study was to examine the characteristics of each activity domain which make up activities of daily living (ADL). We used the ADL index with a unidimensional scale for partially dependent older adults, and examined the distribution of the ADL score and self-evaluation of health and physical fitness. There were 466 subjects from age 60 to 98 (132 male, 81.7 +/- 8.22 years; 334 female, 82.5 +/- 7.25 years). Seventeen items representing 7 domains (holding and changing the posture of the body, dressing, toilet usage, bathing, manual activity, walking, and locomotion and carrying) were selected as ADL items. Each item was measured on a dichotomous scale. A questionnaire consisting of 17 ADL items and two questions concerning self-evaluation of health and physical fitness were administered on subjects by institutional therapists (OT, PT, nurse, social worker). An examination of the cumulative relative frequency distribution curve of total score showed that ADL scores of all domains can be used to assess ADL achievement ability level with a wide range from low to high level. It is inferred that the total score will be low when the score is low for holding and changing body-posture, toilet usage and manual activity. On the other hand, it will be high when the score is high for dressing and walking. ADL abilities for all domains also tended to be higher in persons with higher perceptual levels of health and physical fitness.

[Coronary artery bypass grafting for ischemic heart disease combined with aortitis].

Coronary artery bypass grafting (CABG) was performed in a 67-year-old woman with aortitis. She had a past history of right radical mastectomy. Preoperative coronary angiogram showed diffuse stenotic lesions in both right and left coronary arteries. The pressure gradient between ascending aorta and peripheral radial artery was 90 mmHg and the cause of coronary stenosis seemed to be hypertension due to stenotic distal aorta. The aortogram showed occluded bilateral internal thoracic arteries (ITA) and stenotic abdominal aorta. The cephalic artery was supplied by a large meandering artery via dilated gastroepiploic artery (GEA). And thus the GEA was not useful for CABG. Quadruple CABG was performed with mild varicose saphenous vein (saphenous vein grafts to left anterior descending artery and third branch, and sequential saphenous vein graft to right posterior descending and atrioventricular branches). It seemed to be important to demonstrate the arterial lesion of ITA and GEA before CABG in patients with aortitis.

Generation of intracellular domain of insulin receptor tyrosine kinase by gamma-secretase.

The proteolytic cleavage of a precursor protein into alpha- and beta-subunits by furin is required to form functional insulin receptor (IR). In this study, we examined if IR undergoes the additional presenilin (PS)/gamma-secretase-dependent processing. In cells treated with gamma-secretase inhibitors or expressing the dominant-negative PS1 variant led to the accumulation of an endogenous IR C-terminal fragment. In the presence of proteasome inhibitors, we detected a PS/gamma-secretase cleavage product of the IR, termed the IR intracellular domain (ICD). Cellular fractionation and confocal microscopy analyses showed that the IR-ICD is predominantly detected in the nucleus. These data indicate that IR is a tyrosine kinase receptor, which undergoes PS/gamma-secretase-dependent processing. We also show that the autophosphorylation levels of the IR beta-subunit upon insulin stimulation were decreased by the inactivation of PS/gamma-secretase, raising the possibility that the PS/gamma-secretase proteolysis of IR may play a modulatory role in insulin signaling.

[Experimental scoliosis in the rat spine induced by binding the spinous processes].

The effects of limitation of spinal mobility in forward flexion on the development of scoliosis was studied in experimental animals. The spinous processes of three lumbar vertebrae of young female rats were sutured together to make persistent limitation in the forward flexion of the spine. The lumbar vertebrae of rats, which normally are kyphotic in their profile, frequently became flattened or lordotic after the operation. Scoliosis with a primary curve at the sutured site was produced, although the curve magnitude was mild in 38% of 73 operated rats, and appeared more frequently in those which had more limitation in the spinal mobility at the sutured vertebrae than in those with less limitation. In histological examinations, the coronal sections of vertebral specimens of scoliotic animals demonstrated wedge-shaped intervertebral discs, deviation in the vertebral nucleus pulposus toward the convex side of scoliosis, and a degeneration in the annulus fibrosus. Thickening of the cartilaginous endplates on the convex side and a rupture in them on the concave side were observed in some of the animals. These findings indicated that the experimentally produced scoliosis was structural. This experimental study suggested that a limitation in the spinal mobility in forward flexion has a causative effect on the development and progress of idiopathic scoliosis.

[Isoelectroforetic analysis of alpha 1-acid glycoprotein (alpha 1-AG) in gynecological malignancy].

alpha 1-acid glycoprotein (alpha 1AG) in the blood of gynecologic malignancy was measured by the S.R.I.D. method and analyzed by isoelectrofocusing. The usefulness of these methods for diagnosis and prognosis was examined. A. Quantitative analysis In normal healthy women alpha 1AG was measured (62.2 +/- 2.7 mg/dl) and did not increase before operation in benign gynecologic tumor. In cervical cancer, according to the development of cancer, a quantitative increase was apparent. Upon estimation, a significant difference from healthy women was observed at T1 but not T0. Two weeks after operation a quantitative increase was observed after which there was a gradual decrease but one year later not down close to the value before operation even in patients with good prognosis. In recurrent patients there was a marked increase. In ovarian cancer stage III, alpha 1AG markedly increased to 144.9 +/- 36.6 mg/dl before the operation, being a higher value. B. Isoelectrofocusing analysis Upon isoelectrofocusing, 9 bands were observed between pH3.1 and pH3.5. In normal healthy women I and IX bands were not recognized. In cervical cancer an increase in ratio groups I and II and groups VIII and IX and a decrease in ratio group (IV, V, VI) were seen according to development of the cancer. Ovarian cancer showed the same course as cervical cancer. The measurement and the isoelectrofocusing analysis of alpha 1AG were useful for diagnosis of gynecologic malignancy and its prognosis, and these methods are especially useful in case of ovarian cancer.

Ultrasonic indicators of ureteric reflux in the newborn.

There is controversy over the value of ultrasonic screening for detection of vesicoureteric reflux (VUR) in babies. We scanned 300 newborn babies and identified 53 with a dilated renal pelvis or other minor abnormalities. Eventually, 9 of these were investigated by voiding cystourethrography and 3 (5 kidneys) proved to have VUR exceeding grade II. In 3 babies the reason for cystourethrography was persistent renal pelvis dilation; in 3 it was urinary tract infection; and in 3 it was a new ultrasonic sign observed early in the series--ballooning of the renal pelvis during voiding. Whereas persistent dilation of the renal pelvis was a non-specific indicator (absent in 2 of the 5 affected kidneys and present in 5 of those unaffected), ballooning was consistently seen in all 5 affected renal pelvises. This sign, easily obtained in a baby who has been sleeping, deserves prospective assessment for its value in screening.

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites.

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.