A Simple Automated Method for Detecting Recurrence in High-Grade Glioma.

Our aim was to develop an automated multiparametric MR imaging analysis of routinely acquired imaging sequences to identify areas of focally recurrent high-grade glioma. Data from 141 patients treated with radiation therapy with a diagnosis of high-grade glioma were reviewed. Strict inclusion/exclusion criteria identified a homogeneous cohort of 12 patients with a nodular recurrence of high-grade glioma that was amenable to focal re-irradiation (cohort 1). T1WI, FLAIR, and DWI data were used to create subtraction maps across time points. Linear regression was performed to identify the pattern of change in these 3 imaging sequences that best correlated with recurrence. The ability of these parameters to guide treatment decisions in individual patients was assessed in a separate cohort of 4 patients who were treated with radiosurgery for recurrent high-grade glioma (cohort 2). A leave-one-out analysis of cohort 1 revealed that automated subtraction maps consistently predicted the radiologist-identified area of recurrence (median area under the receiver operating characteristic curve = 0.91). The regression model was tested in preradiosurgery MRI in cohort 2 and identified 8 recurrent lesions. Six lesions were treated with radiosurgery and were controlled on follow-up imaging, but the remaining 2 lesions were not treated and progressed, consistent with the predictions of the model. Multiparametric subtraction maps can predict areas of nodular progression in patients with previously treated high-grade gliomas. This automated method based on routine imaging sequences is a valuable tool to be prospectively validated in subsequent studies of treatment planning and posttreatment surveillance.

Correlation of autophagy type in podocytes with histopathological diagnosis of IgA nephropathy.

OBJECTIVE: IgA nephropathy (IgA-N) frequently leads to progressive renal failure, thus estimation of the degree of progression is important for patient management. Autophagy is a mechanism that facilitates clearance of waste products to preserve renal function. The aim of this study was to assess autophagy in podocytes in children with progressive IgA-N at initial diagnosis by electron microscopy and investigate the relationship between the types of autophagy and severity of the disease. METHODS: Renal biopsies from 16 children with established progressive IgA-N were examined by light and transmission electron microscopy with reference to autophagy types in the podocytes and histopathological diagnosis of IgA-N. RESULTS: Two autophagy types were found. Type I rarely transformed to autophagic vacuoles and did not dissolve, thus possibly impairing cell function. However, type II frequently transformed to autophagosomes and autophagic vacuoles thus facilitating protein and lipid clearance. Of the 16 children studied, 8 (50%) with type I autophagy at initial diagnosis showed focal proliferative glomerulosclerosis (GN) of mild type (3 cases, 37.5%), mild/moderate type (2 cases, 25%) and moderate type (3 cases, 37.5%). In contrast, the remaining 8 children with type II autophagy at initial diagnosis showed focal proliferative GN of mild type in 7 (87.5%) and mild/moderate type in 1 (12.5%) case. CONCLUSION: In IgA-N children, the occurrence of type I autophagy is correlated with histopathologically more progressive disease, possibly reflecting a tendency to a poorer prognosis.

Tacrolimus (FK506) attenuates biphasic cytochrome c release and Bad phosphorylation following transient cerebral ischemia in mice.

Tacrolimus (FK506) has a neuroprotective action on cerebral infarction produced by cerebral ischemia, however, detailed mechanisms underlying this action have not been fully elucidated. We examined temporal profiles of survival-and death-related signals, Bad phosphorylation, release of cytochrome c (cyt.c), activation of caspase 3 and DNA fragmentation in the brain during and after middle cerebral artery occlusion (MCAo) in mice, and then examined the effect of tacrolimus on these signals. C57BL/6J mice were subjected to transient MCAo by intraluminal suture insertion for 60 min. Tacrolimus (1 mg/kg, i.p.) was administered immediately after MCAo. There were biphasic increases in the release of cyt.c in the ischemic core and penumbra; with the first increase toward the end of the occlusion period and the second increase 3-12 h after reperfusion. Tacrolimus significantly inhibited the increase of cytosolic cyt.c during ischemia and reperfusion. Phosphorylated Bad, Ser-136 (P-Bad(136)) and Ser-155 (P-Bad(155)) were detected 30 min after MCAo and after reperfusion in the ischemic cortex, respectively. Tacrolimus increased P-Bad(136) during ischemia and prolonged P-Bad(155) expression after reperfusion. Tacrolimus also decreased caspase-3 and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling-positive cells, and reduced the size of infarct 24 h after reperfusion. Our study provided the first evidence that the neuroprotective action of tacrolimus involved inhibition of biphasic cyt.c release from mitochondria, possibly via up-regulation of Bad phosphorylation at different sites after focal cerebral ischemia and reperfusion.

A functional MRI study of somatotopic representation of somatosensory stimulation in the cerebellum.

Somatotopic representation in the cerebral cortex of somatosensory stimulation has been widely reported, but that in the cerebellum has not. We investigated the latter in the human cerebellum by functional MRI (fMRI). Using a 1.5 tesla imager, we obtained multislice blood oxygen level-dependent fMRI with single-shot gradient-echo echoplanar imaging in seven right-handed volunteers during electrical stimulation of the left index finger and big toe. In the anterior and posterior cerebellum, activated pixels for the index finger were separate from those for the toe. This suggests that somatosensory stimulation of different parts of the body may involve distinct areas of in the cerebellum as well as the cerebral cortex.

Regional N-acetyl-aspartate level and immunohistochemical damage in the hippocampus after transient forebrain ischemia in gerbils.

We investigated changes in regional N-acetyl-aspartate (NAA) levels in the vulnerable CA1 and resistant CA3 areas of the hippocampus after transient forebrain ischemia in gerbils. Under light ether anesthesia, bilateral common carotid arteries of adult male Mongolian gerbils (60-80 g) were occluded for 5 min and reperfused for 7 days. Brains from experimental and control gerbils (n = 4 each) were frozen in situ, and frozen sections (20 microm) were prepared using cryostat (-20 degrees C). After overnight lyophilization, the CA1 and CA3 areas were dissected out separately, weighed (50-200 microg), and the supernatant of the perchloric acid extract was used for assay of NAA using HPLC. Adjacent 10 microm-thick sections were used for immunohistochemical analysis using antiserum against microtubule-associated protein I and II. The preischemic NAA levels were not significantly different between CA1 and CA3 areas. After transient ischemia, a significant (P < 0.01) decrease in the NAA level was observed in the CAI area, but not in the CA3 area of the hippocampus. Immunohistochemical ischemic damage evolved only in the CA1 area. Thus, the decrease of the regional NAA level was associated with development of immunohistochemical neuronal damage.

Comparison of three-dimensional, two-dimensional and color Doppler ultrasound in predicting the presence of a nuchal cord at birth.

OBJECTIVES: To compare the use of two-dimensional, color Doppler and three-dimensional ultrasound for predicting the presence of a nuchal cord at birth. METHODS: Eighty-five singleton pregnancies without nuchal cord and 35 with nuchal cord (30 with a single coil of cord, four with a double coil, and one in which the cord was coiled around the neck three times) were studied within 1 week before delivery using transabdominal three-dimensional sonography. Two-dimensional and color Doppler ultrasound were also conducted for comparison. RESULTS: Two-dimensional, color Doppler and three-dimensional sonography identified in utero 24 (69%), 29 (83%) and 25 (71%), respectively, of the cases of nuchal cord found at birth. There were no significant differences in overall diagnostic indices of each diagnostic modality for detecting nuchal cord. However, the ability to view the nuchal cord (subjective assessment of the ease of visualization of nuchal cord) was better with three-dimensional sonography than with two-dimensional or color Doppler ultrasound. CONCLUSIONS: Three-dimensional surface imaging does not provide more useful diagnostic information compared with two-dimensional and color Doppler ultrasound for detecting nuchal cord in utero.

Morphological differences between glomerular epithelial cells (GEC) excreted during chemotherapy with antineoplastic drugs and GEC excreted in renal diseases.

BACKGROUND: To better understand the mechanisms of glomerular epithelial cell (GEC) injuries in various diseases, we compared GEC excreted during chemotherapy (antineoplastic drugs) and GEC excreted in renal diseases. METHODS: For 19 patients undergoing chemotherapy (85 courses), 69 patients with IgA nephropathy and 16 patients with Henoch-Schölein purpura nephritis, the number of excreted GEC and GEC casts were counted by an immunofluorescent study. The morphological features of GEC were also studied in an immunofluorescent study combined with Hoechst stain. RESULTS: Glomerular epithelial cells were detected in 78% of the chemotherapy courses and in 94% of the patients with renal diseases. The GEC casts were observed in 2% of chemotherapy courses, while in renal diseases GEC casts were observed in 60% of the patients. Proteinuria (>30 mg/dL) and hematuria were not identified in any of the chemotherapy courses. The morphology and size of GEC were more variable than that in patients with nephropathy. Furthermore, GEC in patients undergoing chemotherapy often showed small nuclei and fragmented nuclei, which were rarely observed in patients with nephropathy. CONCLUSIONS: These results showed that the detachment of podocytes was not directly associated with proteinuria or hematuria. The findings also suggest that GEC are damaged via an apoptotic process by chemotherapy. On the contrary, GEC may be detached through a non-apoptotic process in renal diseases.

Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.

Although accumulating evidence indicates that cAMP response element-binding protein (CREB) phosphorylation mediates not only synaptic plasticity but also survival of certain neurons, it remains uncertain whether CREB phosphorylation induced after metabolic insult leads to CRE-mediated gene transcription and is involved in cell survival or not. In the present study, we clarified that (1) CREB phosphorylation and ischemic tolerance induced after preconditioning ischemia in the hippocampal neurons was abolished by MK801 administration in gerbil global ischemia model, (2) CREB phosphorylation induced after exposure to glutamate in cultured neurons was inhibited by removal of extracellular calcium, by MK801 and by an inhibitor of calcium-calmodulin-dependent protein kinase (CaMK) II and IV, (3) inhibitor of CaMK II-IV or CRE-decoy oligonucleotide suppressed upregulation of BCL-2 expression and accelerated neuronal damage after exposure to glutamate, and (4) CREB phosphorylation induced in the hippocampal neurons after ischemia and in cultured neurons after exposure to glutamate was followed by CRE-mediated gene transcription in transgenic mice with a CRE-LacZ reporter. Our results suggest that CREB phosphorylation in neurons after ischemia and exposure to glutamate is induced by NMDA receptor-gated calcium influx and subsequent activation of CaMK II-IV and that CREB phosphorylation after metabolic stress might show a neuroprotective response through CRE-mediated gene induction.

Influence of prolonged corticosteroid therapy on the outcome of steroid-responsive nephrotic syndrome.

Eighty-six patients (59 males and 27 females) diagnosed with steroid-responsive nephrotic syndrome during childhood were identified. The patients were 20-40 years of age (mean 27.0 +/- 5.0) with a mean follow-up period of 19.5 +/- 5.9 years. All patients had been treated with a long-term tapering corticosteroid therapy. Thirty patients had also received a course of cyclophosphamide (2 mg/kg/day for 12 weeks). Sixty-six had achieved sustained remission off corticosteroids, while 20 were still receiving corticosteroids to maintain remission. None of the 86 patients had proteinuria or renal insufficiency at the time of the study. Mean final heights in males and females were similar (-0.51 +/- 1.21 and -0.23 +/- 1.16 standard deviation score). Mean final height of 20 steroid-dependent patients was significantly less than that of 66 in remission off corticosteroids (p < 0.005). Ten cyclophosphamide-treated patients got married and 9 had at least 1 healthy child. In children with steroid-responsive nephrotic syndrome, the need for corticosteroid therapy to maintain remission may be associated with decreased adult height. Patients who received a 12-week course of cyclophosphamide are likely to be normally fertile as adults.

A potent AMPA/kainate receptor antagonist, YM90K, attenuates the loss of N-acetylaspartate in the hippocampal CA1 area after transient unilateral forebrain ischemia in gerbils.

By analyzing histological damages and the regional N-acetylaspartate (NAA) level simultaneously, we evaluated the effect of an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist, YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3-(1H,4H)-quinoxalinedione monohydrochloride], in unilateral forebrain ischemia in gerbils. The right common carotid artery was clipped for 5 min under ether anesthesia, and reperfused for 7 days. The frozen brain sections were lyophilized and the hippocampal CA1 area was dissected out for HPLC assay of NAA. An adjacent section was stained with hematoxylin-eosin for counting survived neurons per 1 mm pyramidal layer of the hippocampal CA1 area. Postischemic administration of YM90K at 20 mg/kg and 25 mg/kg attenuated the decrease of both the number of survived neurons and the NAA level on the ischemic side in a dose-dependent manner. A significant linear correlation was observed between the NAA level and the number of intact neurons. These results indicated that the NAA level could be used as an index of neuroprotective effects of pharmacological agents in global cerebral ischemia.

Urinary podocytes in primary focal segmental glomerulosclerosis.

BACKGROUND/AIM: Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome. Although the pathogenesis is not known, recent studies suggest that FSGS may be a podocyte disease. The aim of this study was to look for podocyte injury in this disease, using measurements of urinary podocytes. METHODS: We examined the first morning urine of the day collected from 71 patients (45 men and 26 women, median age and range 11.2 and 3-29 years) diagnosed as having nephrotic syndrome. Freshly voided urine samples were examined by immunofluorescence labeling using monoclonal antibodies against human podocalyxin. Renal histological examinations were performed in 58 of the 71 patients: 28 had minimal-change disease, 20 had FSGS, and 10 had membranous nephropathy. RESULTS: Median and range of urinary podocytes measured were 0.2 and 0-40.8 cells/ml for 71 patients with nephrotic syndrome and 0 and 0-0.8 cells/ml for normal healthy control subjects (n = 200). Patients with FSGS had significantly higher levels of urinary podocytes (median and range 1.3 and 0-40.8 cells/ml) than those with minimal-change disease (median and range 0 and 0-6.9 cells/m; p = 0.003) or membranous nephropathy (median and range 0 and 0-1.4 cells/ml; p = 0.02). CONCLUSIONS: The urinary excretion of podocytes is significantly higher in patients with FSGS as compared with those having membranous nephropathy or minimal-change disease. These findings suggest that podocyte injury and loss in the urine may have an important role in the pathogenesis of FSGS.

Decrease in N-acetylaspartate/creatine ratio in the motor area and the frontal lobe in amyotrophic lateral sclerosis.

We studied whether N-acetylaspartate (NAA), a neuronal marker, is reduced in the brain of 14 patients with clinically definite amyotrophic lateral sclerosis (ALS) and whether NAA levels in the motor area and frontal lobe correlate with the clinical features, including frontal lobe function. We also studied 14 normal controls were evaluated. We obtained peak integrals in 1H magnetic resonance spectroscopy (MRS) for NAA, creatine (Cr), and choline-containing compounds (Cho). Severity of the disease was determined using the manual muscle strength test, and the Norris limb and bulbar scales. In the patients, the NAA/Cr ratio was reduced in the motor area and frontal lobe, while the Cho/Cr ratio was normal throughout the brain. There were significant correlations between the NAA/Cr ratio in the motor area and the Norris limb scale (r = 0.50; P < 0.01) and between the NAA/Cr ratio in the frontal lobe and the number of categories achieved in the Wisconsin Card Sorting test (r = 0.71; P < 0.05), implying frontal lobe dysfunction. These correlations suggest that a reduced NAA/Cr ratio is a marker of cortical neuronal loss and dysfunction in ALS.

Glomerular CD8+ cells predict progression of childhood IgA nephropathy.

The aim of this study was to evaluate whether the infiltrating T-lymphocyte can be a predictor in the disease progression of IgA nephropathy (IgAN). Twenty children with IgAN, followed for more than 5 years, were divided into progressive (n=5) and non-progressive groups (n=15). We assessed glomerular and interstitial infiltration of T-lymphocytes (CD4+ and CD8+ cells) and expression of alpha-smooth muscle actin (alpha-SMA) and transforming growth factor-beta (TGF-beta) using an indirect immunofluorescence method on the renal biopsies. We analyzed their relationship to the degree of proteinuria, histological changes, and prognosis. The number of CD8+ cells in glomeruli and in interstitium was higher in the progressive group than in the non-progressive group. The glomerular alpha-SMA staining was more intensive in the progressive group than in the non-progressive group. Urinary protein and the degree of histological changes were also higher in the progressive group than in the non-progressive group. Among these markers, the number of glomerular CD8+ cells was the most apparent difference between the two groups. In conclusion, these results indicate that the number of glomerular CD8+ cells is the most sensitive predictor of disease progression in childhood IgAN.

[Strip immunoblot assay (SIA) with recombinant antigens and synthetic peptide for detection of anti HIV-1 and HIV-2 antibodies].

The serodiagnosis of human immunodeficiency virus (HIV) infection has widely been established by the screening test and the confirmatory test. At present, Western blot (WB) assay is mostly used as the confirmatory test. However, this method has the problem in that the sensitivity and the specificity are not enough. A new confirmatory test "CHIRON RIBA HIV-1/HIV-2 SIA" developed by Chiron Corporation uses an immunoblot enzyme immunoassay technique for detection of anti HIV-1 and/or HIV-2 antibodies. This assay employs four recombinant viral antigens (gp120, gp41, p24/p26 and p31) and a synthetic viral antigen (HIV-2 envelope peptide). The characteristic of this method is that the HIV-1 infection and the HIV-2 infection can be differentiated from each other. We therefore compared this SIA method with the WB1 assay for detection of anti HIV-1 antibodies and with the WB2 assay for detection of anti HIV-2 antibodies. Eighty samples from normal adults without HIV infection and known to be negative by three HIV screening tests, respectively, were tested by SIA, WB1 and WB2 assays. The negative rates (specificities) were 97.5%, 80.0% and 87.5% by the SIA, WB1 assay and WB2 assay, respectively. With forty samples from patients without HIV infection but known to be positive by at least one HIV screening test, the negative rates (specificities) were 97.5%, 72.5% and 85.5% by the SIA, WB1 assay and WB2 assay, respectively. The results indicated that the SIA method was more specific than two WB assays. Forty samples from patients with HIV-1 infection and known to be positive by three HIV screening tests, were tested by the SIA and WB1 assay. The positive rates (sensitivities) were 97.5% and 75.0% by the SIA and WB1 assay, respectively. With thirteen samples from patients with HIV-2 infection and known to be positive by three HIV screening test, the positive rates (sensitivities) were 100% and 92.3% by the SIA and WB1 assay, respectively. The results indicated that the SIA method was more sensitive than the WB1 assay. Three sets of sera, which were collected during seroconversion for HIV-1 antibody, were used to compare the positive readings by the SIA and WB1 assay. The SIA method indicated the positive readings earlier than the WB1 assay. The present findings indicated that the SIA method was more specific and sensitive than the WB assay, and would be useful as a confirmatory test.