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Introduction: USA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan., Methods: We obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors., Results: Twenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores., Conclusions: Our results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

After traumatic spinal cord injury (SCI), a scar may form with a fibrotic core (fibrotic scar) and surrounding reactive astrocytes (glial scar) at the lesion site. The scar tissue is considered a major obstacle preventing regeneration both as a physical barrier and as a source for secretion of inhibitors of axonal regeneration. Understanding the mechanism of scar formation and how to control it may lead to effective SCI therapies. Using a compression-SCI model on adult transgenic mice, we demonstrate that the canonical Wnt/β-catenin signaling reporter TOPgal (TCF/Lef1-lacZ) positive cells appeared at the lesion site by 5 days, peaked on 7 days, and diminished by 14 days post injury. Using various representative cell lineage markers, we demonstrate that, these transiently TOPgal positive cells are a group of Fibronectin(+);GFAP(-) fibroblast-like cells in the core scar region. Some of them are proliferative. These results indicate that Wnt/β-catenin signaling may play a key role in fibrotic scar formation after traumatic spinal cord injury., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: In daily clinical work, we often face delayed or recurrent pneumothorax after radiofrequency (RF) ablation for lung tumors, but a large study on this theme has not been done. Thus, we examined the rate of delayed or recurrent pneumothorax after RF ablation for lung tumors and the risk factors associated with its occurrence., Materials and Methods: This retrospective study was based on 194 consecutive sessions of percutaneous RF ablation of 220 lung tumors in 68 patients performed under CT fluoroscopic guidance at a single institution. Numerous variables were analyzed to the assess risk factors for delayed or recurrent pneumothorax., Results: Pneumothorax after RF ablation occurred in 82 of 194 ablation sessions (42.3%). Thirty-three of 82 sessions had either delayed pneumothorax (n = 20) or recurrent pneumothorax (n = 13). The other 49 sessions had nonprogressive pneumothorax. Only contact of the ground-glass opacity (GGO) that emerged around the ablated lesion with the pleura significantly correlated with the frequency of delayed or recurrent pneumothorax in comparisons between no pneumothorax vs delayed/recurrent pneumothorax and between nonprogressive pneumothorax vs delayed/recurrent pneumothorax. The mean (+/- SD) duration before confirmation of the presence or recurrence of pneumothorax was 24.0 +/- 66.4 h. Among the 33 sessions with delayed or recurrent pneumothorax, 4 subjects needed additional treatment., Conclusion: Our results indicated that delayed or recurrent pneumothorax is relatively frequently encountered after RF ablation of lung tumors. Particular care must be taken with regard to the occurrence of delayed or recurrent pneumothorax when contact of a GGO with the pleura is seen after RF ablation.

Objectives: This study was initiated to examine if exposure to cadmium (Cd) was high also outside of the previously identified Itai-itai disease endemic region in the Jinzu River basin in Toyama prefecture in Japan., Methods: Morning spot urine samples were collected in June-August 2004 from 651 adult women (including 535 never-smokers) in various regions in Toyama prefecture, and subjected to urinalyses for cadmium (Cd), alpha1-microglobulin (alpha1-MG), beta2-microglobulin (beta2-MG), N-acetyl-beta-D-glucosaminidase (NAG), specific gravity (SG or sg) and creatinine (CR or cr). Three months later, the second urine samples were collected from those with elevated Cd in urine (e.g., > or =4 microg/g cr), together with answers to questionnaires on shellfish consumption., Results: The geometric mean (GM) Cd, alpha1-MG, beta2-MG and NAG (after correction for CR) for the total participants were 2.0 microg/g cr, 2.4 mg/g cr, 104 microg/g cr and 2.8 units/g cr, respectively; further analysis with never-smoking cases only did not induce significant changes in these parameters. Analyses of the second urine samples from the high Cd subjects showed that there was substantial decrease (to about a half) in Cd in the 3-month period, and that the decrease was accompanied by reduction in alpha1-MG and NAG (beta2-MG did not show elevation even in the first samples). The urinalysis results in combination with the results of the questionnaire survey suggest that the high urinary Cd was temporary and might be induced by intake of shellfish that is edible whole., Conclusions: The overall findings appear to suggest that Cd exposure in Toyama populations (outside of the Itai-itai disease endemic region) was at the levels commonly observed on the coast of the Sea of Japan, and that the Cd level in urine might be modified by the intake of some types of seafood. Further studies are necessary to elucidate the relation of urinary Cd with seafood intake.

Canine pigmented epidermal nevus (CPEN) is a skin disorder of some breeds of dog characterized by multiple black plaques of the haired and non-haired skin. Three cases of pigmented cutaneous papillomatosis (previously described also as CPEN) in pug dogs were investigated histopathologically, immunohistochemically and electron microscopically. Additionally, DNA analyses with the polymerase chain reaction (PCR) were performed in two cases. Many nuclei of the stratum granulosa were diffusely immunolabelled for specific structural antigens of bovine papillomavirus (subgroup A), but nuclear inclusion bodies were not detected by retrospective examination of haematoxylin and eosin-stained sections of the affected skin. Aggregates of small numbers of viral particles (ranging from 37 to 43 nm in diameter) with a hexagonal structure were sparsely scattered throughout the nuclei of some of the superficial keratinocytes. PCR amplification targeted for the L1 gene of papillomavirus cloned from a case of CPEN yielded an expected fragment of 194-bp in the two CPEN cases examined but not in a case of canine oral papilloma.

Objectives: The present study was performed to evaluate the efficacy and safety of gantry tilting for the performance of lung biopsy of peripheral small lesions located beneath the rib., Design: Interventional., Materials and Methods: Our study was based on 22 of 237 lesions for which percutaneous needle biopsies of the lung were performed under CT scan-fluoroscopic guidance at our institution between January 2000 and August 2002. For these 22 lesions, a biopsy was performed with gantry tilt because a rib blocked the biopsy route even after trials to change the relationship between the target and the rib. The characteristics of each lesion, the success rate for obtaining an adequate specimen, and the ability to determine whether the lesion was malignant or benign were investigated, specific cell types were characterized, and the complications that were encountered were identified., Results: In all 22 lesions, adequate specimens for cytopathologic evaluation were obtained using fine-needle aspiration biopsy, tissue core biopsies, or both. In 21 lesions, whether the lesion was malignant or benign was precisely diagnosed, and in 19 lesions the specific cell type was determined. No serious complications occurred., Conclusion: Percutaneous needle biopsy under CT scan-fluoroscopic guidance with gantry tilt is a useful and safe technique for the biopsy of small lung nodules located beneath the rib.

A retrospective study of 3102 injured snowboarders from 1992 to 1999 was undertaken using the medical records and questionnaires the patients had been asked to complete at the time of consultation. These cases represent all the injuries with medical treatment that occurred in five skiing and snowboarding resorts in the Nagano area. The total number of the injuries was 3243. The number of patients each year rapidly increased over the eight seasons. In descending order, the injuries were seen in the head and face, left upper extremity, spine, chest and abdomen, left lower extremity, right upper extremity, and right lower extremity. The number of head and face injuries was 829 (25%) followed by 768 (24%) left upper extremity injuries. With regard to the type of injury, fracture was the most frequent injury. There were 998 fractures, of which 625 were fractures of the upper extremity. Contusions were frequent on the head, face, chest, abdomen, and pelvis. Lacerations were frequent on the head and face. Sprains were frequent in the lower extremities, and dislocations were seen frequently in the upper extremities. The sharp increase in the number of patients was thought to reflect the increase in the population of snowboarders in Japan. The reason for the high concentration of injuries to the head and upper extremity is thought to derive from the characteristic riding form of snowboarding, with both legs fixed to one short, wide board. To decrease the injuries and to develop snowboarding as a safe and exciting sport, snowboarders, instructors, slope managers, and equipment manufacturers must comprehend the patterns of snowboarding injuries and cooperate to devise preventive measures., (The Japanese Orthopaedic Association)

Objectives: The goal of our study was to evaluate the efficacy and safety of a new type of automated cutting needle for tissue-core lung biopsy under real-time CT fluoroscopic guidance., Design: Observational, Materials and Methods: One hundred ten percutaneous needle tissue-core lung biopsies were performed with the Auto Surecut needle (Create Medic; Yokohama, Japan) under CT fluoroscopic guidance, and the specimens obtained underwent histopathologic evaluation. A final diagnosis was confirmed by independent surgical pathology., Results: The rate of success for the diagnosis for specimens that were adequate for histopathologic analysis was 94.5% (104 of 110 specimens). The sensitivity, specificity, and accuracy in diagnosing malignancy were 95.1%, 100%, and 96.2%, respectively. A specific cell type could be characterized in 95.2% of those 104 lesions (99 lesions; malignant, 76 lesions; benign, 23 lesions). The specific cell type was precisely diagnosed, and was confirmed after surgery in 65 malignant lesions and 23 benign lesions. The biopsy-induced complications encountered were pneumothorax in 34.5% (38 of 110 patients) and hemoptysis in 6.4% (7 of 110 patients). No patient had a serious complication., Conclusion: CT fluoroscopy-guided lung biopsy using the Auto Surecut needle provides a high degree of diagnostic accuracy, allows for the specific characterization of lung nodules, and can be performed safely.

Wilms tumor gene WT1 is expressed at high levels in hematopoietic malignancies, such as leukemias and myelodysplastic syndromes (MDS), and in various kinds of solid tumors, including lung cancer, and it exerts an oncogenic function in these malignancies. IgM and IgG WT1 antibodies were measured by means of dot blot assay in 73 patients with hematopoietic malignancies (16 acute myeloid leukemia [AML], 11 acute lymphoid leukemia [ALL], 13 chronic myeloid leukemia [CML], and 33 MDS) and 43 healthy volunteers. Immunoglobulin IgM, IgG, and IgM+IgG WT1 antibodies were detected in 40 (54.8%), 40 (54.8%), and 24 (32.8%), respectively, of the 73 patients with hematopoietic malignancies, whereas 7 (16.2%), 2 (4.7%), and none of the 43 healthy volunteers had IgM, IgG, or IgM+IgG WT1 antibodies, respectively. Furthermore, immunoglobulin isotype class switching of WT1 antibodies from IgM to IgG occurred in conjunction with disease progression from refractory anemia (RA) to RA with excess of blasts (RAEB), and further to RAEB in transformation (RAEB-t) in MDS patients. These results showed that humoral immune responses against the WT1 protein could be elicited in patients with WT1-expressing hematopoietic malignancies, and they suggested that the helper T-cell responses needed to induce humoral immune responses and immunoglobulin isotype class switching from IgM to IgG were also generated in these patients. Our findings may provide new insight into the rationale for elicitation of cytotoxic T-cell responses against the WT1 protein in cancer immunotherapy using the WT1 vaccine.

Objectives: To evaluate the efficacy of simple aspiration of air from the pleural space to prevent increased pneumothorax and to avoid chest tube placement in cases of pneumothorax following CT-guided lung biopsy., Design: Observational., Materials and Methods: One hundred thirty-four consecutive percutaneous needle lung biopsies using real-time CT fluoroscopy guidance formed the basis of our study. All patients that demonstrated moderate or severe pneumothorax on postbiopsy chest CT images underwent percutaneous manual aspiration regardless of symptoms while on the CT scanner table. Correlation between the incidence of pneumothorax after biopsy and many factors (i.e., gender, age, number of pleural passes, presence of emphysema, lesion size, and lesion depth) were determined, and management of each case of biopsy-induced pneumothorax was reviewed., Results: Postbiopsy pneumothorax occurred in 46 of 134 procedures (34.3%). Twenty of the 46 patients were treated by manual aspiration, while 26 patients were simply observed. In 43 of the 46 pneumothoraces (93.5%), the pneumothorax resolved completely on follow-up chest radiographs without requiring tube placement. Only three patients (2.2% of the entire series; 6.5% of those who had pneumothorax develop) required chest tube placement. The risk of pneumothorax significantly increased with lesion size and depth., Conclusion: Results of our nonprospective, nonrandomized study suggest that percutaneous manual aspiration of biopsy-induced pneumothorax performed immediately after biopsy may prevent progressive pneumothorax and subsequent chest tube placement.

A water-soluble Touchi-extract (TE) exerts a potent inhibitory activity against rat intestinal alpha-glucosidase in foodstuffs, and elicited anti-glycemic effects in rats and humans with single-bolus oral administration. In the present study, genetically modified diabetic model KKAy mice were used to examine the long-term effects of TE. Eight-week-old male KKAy mice were fed with CE-2 chow containing 0.08 and 0.4% of TE for 60 days. In the latter group, fasting blood glucose levels decreased (6.68 +/- 0.41 mmol/L) significantly (p<0.05) after a 60-day ingestion period compared with controls (8.75 +/- 0.54 mmol/L). Moreover, postprandial blood glucose levels were also significantly reduced (16.79 +/- 2.28 mmol/L; p<0.01) after ingesting TE for only 30 days compared with controls (28.49 +/- 0.59 mmol/L). On oral TE treatment for 60 days, postprandial increases in the blood glucose level after oral loading of sucrose (2 g/kg) at 30 (p<0.05) and 60 (p<0.01) min were significantly depressed compared with controls. Indexes for serum lipids; viz., total cholesterol (p<0.05) and triglyceride (p<0.01) levels significantly decreased after TE ingestion. Indexes for hepatic functions, such as glutamic-oxaloacetic transaminase (p<0.01), glutamic-pyruvic transaminase and gama-glutamyl transpeptide levels, were similarly suppressed. Organ weights of the heart, kidney, jejunum, liver and spleen increased in control KKAy mice due to hyperinsulinemia. Interestingly, the respective organ weights decreased (p<0.05, 0.01) and the jejunum length was reduced (p<0.05) significantly in the TE-treated groups. All in all, TE demonstrated an anti-hyperglycemic effect and may have potential use in the management of non-insulin-dependent diabetic mellitus.

Water-extracted Touchi, a traditional Chinese food, exerts a strong inhibitory activity against rat intestinal alpha-glucosidase in foodstuffs, and Touchi-extract (TE) has been shown to have an antihyperglycemic effect in rats and humans after a single oral administration. In the present complementary study, the effects of powdered Houji-tea with or without (placebo) TE, a formula designed to enhance good compliance, were monitored in a 3-mo double-blind randomized group comparison study with placebo controls in humans with borderline and mild type-2 diabetes (n = 36). All subjects ingested Houji-tea with or without 0.3 g of TE before each of three meals per day for 3 mo. In the TE group, initial fasting blood glucose (6.9 +/- 0.1 mmol/L) and glycated hemoglobin (HbA(1c); 6.1 +/- 0.1%) levels gradually decreased; fasting blood glucose decreased significantly after 3 mo (6.4 +/- 0.3 mmol/L; P < 0.05) as did HbA(1c) (5.6 +/- 0.2%; P < 0.01) levels at 2 mo postingestion of TE and thereafter. In contrast, fasting blood glucose and HbA(1c) levels did not change in the placebo group. In this study, other biochemical variables were not affected in any of the subjects, and no one complained of any side effects or abdominal distension. Moreover, there was no deterioration as assessed by fasting blood glucose and HbA(1c) levels after withdrawal of TE ingestion. Thus, the alpha-glucosidase inhibitory TE demonstrated an antihyperglycemic effect and may prove useful for improving glycemic control in subjects suffering from borderline and type-2 diabetes mellitus.

A water-soluble extract of Touchi, a traditional Chinese food, was found to exert a strong inhibitory activity against rat intestinal alpha-glucosidase. We orally administered sucrose (2 g/kg) with or without Touchi extract (TE) to normal rats at 100 and 500 mg/kg. Postprandial increases in blood glucose levels at 30 and 60 min after the administration of TE were significantly depressed compared with controls. In humans, eight borderline diabetic subjects were administered 0.1-10.0 g TE before sucrose loading (75 g). TE decreased the glycemic response dose dependently after sucrose loading. Compared with the area under the curve of the postprandial rise in blood glucose with various doses, TE elicited a significant antiglycemic effect at a minimum effective dose of 0.3 g. In addition, when four diabetics were administered 0.3 g TE before eating 200 g of cooked rice, the postprandial increases in blood glucose and mean insulin levels were significantly depressed at 60 and 120 min, respectively, after ingestion compared with levels when no TE was administered. TE, which exhibits alpha-glucosidase inhibitory activity, demonstrated an antihyperglycemic effect and may have potential use in the management of patients with non-insulin-dependent diabetic mellitus.

A method for the rapid trace analysis of 17 residual pesticides in water by narrow-bore capillary (I.D. 100 microm) gas chromatography-mass spectrometry (GC-MS) using a programmable temperature vaporizer (PTV) was discussed. The method consisted of a large-volume injection (40 microl) by a PTV, high-speed analysis using a narrow-bore capillary column and MS detection. The PTV with solvent vent mode was very useful for large-volume injection into a narrow-bore capillary column because the injected solvent volume could be reduced to less than 2 microl. The analysis time was 8.5 min [less than 50% of the analysis time using conventional columns (I.D. 250 microm)]. A 10-ml volume of river water was extracted by dichloromethane (4 ml), and then the extract was condensed to 1 ml. This extract was analyzed. Mean recoveries for river water spiked at 100 pg/ml ranged from 83.4 to 96.7%. The limit of detections of the 17 pesticides ranged from 1 to 100 pg/ml.

Background: Hepatocellular carcinoma (HCC) commonly develops in patients with chronic hepatitis or cirrhosis of the liver caused by hepatitis-C-virus (HCV) infection. We prospectively studied whether irregular regeneration of hepatocytes is a risk factor for HCC in these patients., Methods: 242 patients were enrolled after liver biopsy and followed up by ultrasonographic scanning every 3 months. We examined age, sex, platelet count, the diagnosis of cirrhosis or chronic hepatitis, liver-cell dysplasia, and irregular regeneration. We classified irregular regeneration as slight or severe, based on histological expression of pleiomorphism, anisocytosis, bulging, and map-like distribution of hepatocytes., Findings: 37 of 63 patients with cirrhosis and 26 of 179 with chronic hepatitis were judged to have severe irregular regeneration. HCC was diagnosed in 33 of 63 patients with cirrhosis (29 had severe irregular regeneration) and 12 of 179 patients with chronic hepatitis (11 had severe irregular regeneration) during mean follow-up of 5.5 years (SD 4.1; range 1-16). Multivariate analysis with a proportional-hazards model showed severe irregular regeneration (relative risk 15.1 [95% CI 5.6-40.7], p<0.0001) and a diagnosis of cirrhosis (3.8 [1.7-8.2], p=0.0008) to be significant risk factors for HCC. Within the diagnostic categories, irregular regeneration was also significant (cirrhosis 6.8 [2.1-21.9], p=0.0014; chronic hepatitis 28.5 [2.9-276.4], p=0.0038)., Interpretation: We recommend that liver biopsy to look for irregular regeneration should be done in patients with HCV-related chronic liver diseases. Those with severe irregular regeneration should be followed up carefully.

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34- cell populations, and the CD34+ cells into nine subsets (CD34+ CD33-, CD34+ CD33+, CD34+ CD38-, CD34+ CD38+, CD34+ HLA-DR-, CD34+ HLA-DR+, CD34+ c-kit(high), CD34+ c-kit(low), and CD34+ c-kit-) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+ CD33-, CD34+ CD33+, CD34- CD33+, and CD34- CD33-). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34- cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 x 10(-2)) or undetectable (< 10(-2)) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 x 10(-1). Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

Thirty-one patients (27 with acute myeloid leukemia [AML], 2 with acute lymphocytic leukemia [ALL], and 2 with acute mixed lineage leukemia [AMLL]) treated with conventional chemotherapy (CHT) and 23 patients (13 AML, 5 ALL, and 5 with chronic myeloid leukemia [CML]) treated with allogeneic bone marrow transplantation (BMT) were monitored for WT1 expression levels in BM and peripheral blood (PB) by reverse transcriptase-polymerase chain reaction over a long-term period (mean, 29 months for CHT and 24 months for BMT). Sixteen of the patients in the CHT group and 3 in the BMT group who had achieved complete remission suffered clinical relapse. In 10 of these patients, WT1 expression that had returned to normal BM levels (< 10(-3); the WT1 expression level of K562 cells was defined as 1.0) after complete remission (CR) either gradually or rapidly increased again to abnormal levels 1 to 18 months (mean, 7 months) before clinical relapse became apparent. In another 9 patients, WT1 expression never returned to normal BM levels even after CR and the subsequent relapse was accompanied by a rapid increase in WT1 expression to levels higher than 10(-2) (10(-3) levels in PB). On the other hand, the remaining 35 patients (15 CHT and 20 BMT) maintained their CR. In 29 of these patients (11 CHT and 18 BMT), WT1 expression either gradually or rapidly decreased to normal BM levels, whereas in the other 6 (4 CHT and 2 BMT), low or very low levels of WT1 mRNAs (10(-3) to 10(-2) in BM and 10(-5) to 10(-3) in PB) remain detectable, but without any clinical signs of relapse. A clear correlation was found to exist between the minimal residual disease (MRD) detected in the paired BM and PB samples for all types of leukemias (AML, ALL, and CML), with MRD in PB being approximately one-tenth of that in BM. WT1 quantitation of 168 paired BM and PB samples showed that PB samples were superior to BM samples for the detection of MRD. We conclude that monitoring of WT1 expression levels in BM and PB makes it possible to rapidly assess the effectiveness of individual treatment and diagnose clinical relapse in the early stage for all leukemia patients regardless of the presence or absence of tumor-specific DNA markers.

We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5' cap sites of the WT1 gene to the 3' end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 protein levels. These results show that WT1 plays an important role in leukemogenesis.

The breakpoint of 14q32 translocations found in B-cell malignancies was delineated specifically in both metaphase spreads and interphase nuclei by double-color fluorescence in situ hybridization (FISH) using bacteriophage clones containing the human immunoglobulin gamma chain gene locus (Ig gamma) and a cosmid clone, CY24-68, containing VH segments. CY24-68 is more telomeric than Ig gamma, separated by approximately 1 megabase (Mb). FISH studies were performed on four patients with non-Hodgkin's lymphoma (NHL), one with acute lymphoblastic leukemia (ALL), one with plasma cell leukemia (PCL), and three cell lines. In each patient with t(8;14), t(14;18), and t(3;14), the signal of Ig gamma gene was observed on der(14) and that of CY24-68 at respective partner sites of these translocations, 8q24.1, 18q21.3, and 3q27. Interphase nuclei with a signal of Ig gamma clearly separated from that of CY24-68 were more frequently encountered in all of the patients (45% to 74%) than those in normal controls (4% to 5%). Even in cases where only interphase nuclei were available for FISH studies, 14q32 translocations are detected as shown in two patients each with NHL and t(11;14)-carrying PCL. In two cell lines, HS-1 derived from ALL carrying t(8;14) and FR4 derived from a plasmacytoma carrying a complex form of t(8;14), the signal of Ig gamma was observed at the breakpoint region 8q24.1 of the der(8) in addition to the der(14), indicating that translocation event occurred within the Ig gamma locus. Intense Ig gamma signal was found at the breakpoint region on the der(14)t(11;14) in HBL-2 derived from NHL, indicating amplification of the Ig gamma gene, and presumably the resultant chimeric DNA between Ig gamma and DNA sequences at 11q13. The present approach allowed us to unequivocally detect tumor-specific breakpoints of 14q32 translocations. Furthermore, interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies.

The WT1 gene encoding a zinc finger polypeptide is a tumor suppressor gene that plays a key role in the carcinogenesis of Wilms' tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine relative levels of WT1 gene expression (defined in K562 cells as 1.00) in 45 patients with acute myelogenous leukemia (AML), 22 with acute lymphocytic leukemia (ALL), 6 with acute mixed lineage leukemia (AMLL), 23 with chronic myelogenous leukemia (CML), and 24 with non-Hodgkin's lymphoma. Significant levels of WT1 gene were expressed in all leukemia patients and for CML the levels increased as the clinical phase progressed. In striking contrast with acute leukemia, the levels of WT1 gene expression for NHL were significantly lower or even undetectable. Clear correlation was observed between the relative levels of WT1 gene expression (< 0.6 v > or = 0.6) and the prognosis for acute leukemia (AML, ALL, and AMLL). Patients with less than 0.6 levels had significantly higher rates of complete remission (CR), disease-free survival, and overall survival than those with > or = 0.6 levels, whereas CR could not be induced in any of the 7 patients with acute leukemia having greater than 1.0 levels of WT1 gene expression. The quantitation of the WT1 gene expression made it possible to detect minimal residual disease (MRD) in acute leukemia regardless of the presence or absence of tumor-specific DNA markers. Continuous monitoring of the WT1 mRNA was performed for 9 patients with acute leukemia. In 4 patients, MRD was detected 2 to 8 months before clinical relapse became apparent. In 2 other patients, the WT1 mRNA gradually increased after discontinuation of chemotherapy. No MRD was detected in the remaining 3 patients with AML who received intensive induction and consolidation therapy. Simultaneous monitoring of MRD by RT-PCR using primers for specific DNA markers in 3 patients (2 AML-M3 with PML/RAR alpha, and 1 AML-M2 with AML1/ETO) among these 9 patients detected MRD comparable with that obtained from quantitation of WT1 gene expression. In a patient with acute promyelocytic leukemia, the limits of leukemic cell detection by RT-PCR using either WT1 or promyelocytic leukemia/retinoic acid receptor-alpha gene primers were 10(-3) to 10(-4) and 10(-4) for bone marrow, and 10(-5) and 10(-4) for peripheral blood, respectively. Therefore, we conclude that WT1 is a new prognostic factor and a new marker for the detection of MRD in acute leukemia.

Expression patterns of interleukin-6 (IL-6), IL-6 receptor (IL-6R), and gp130 genes in 39 patients with acute myeloid leukemia (AML), in 23 patients with acute lymphoblastic leukemia (ALL), and in 7 patients with acute mixed lineage leukemia (AMLL) were studied by quantitative reverse transcriptase-polymerase chain reaction. Significant levels of IL-6 were expressed in 8 (21%) of 39 AML patients and in 2 (29%) of 7 AMLL patients, whereas in ALL, the expression of IL-6 was almost negligible. IL-6R was expressed in all patients with AML and AMLL, whereas only half of ALL patients expressed low levels of IL-6R as compared with those with AML and AMLL. However, gp130 was ubiquitously expressed in all the leukemia patients, and there was no significant difference in gp130 expression among AML, ALL, and AMLL. Significant correlation was observed between the expression of IL-6R and gp130 in AML. When tested for in vitro response to IL-6, the leukemic cells from 3 of 7 AML, none of 3 ALL, and both of 2 AMLL patients significantly responded to IL-6, showing the correlation between the expression levels of IL-6R and gp130 and the responsiveness of leukemic cells to IL-6. These results showed that quantitation of IL-6R and gp130 expression by reverse transcriptase-polymerase chain reaction is useful for the rapid prediction of the responsiveness of leukemic cells to IL-6, especially in cases of administration of IL-6.

The sensitivity of scid fibroblasts established from C.B17-scid/scid fetuses to the DNA-damaging agents bleomycin, neocarzinostatin, mechlorethamine, mitomycin C, methyl methanesulfonate, and ultraviolet light, all of which induce different types of DNA damage, was examined. Scid fibroblasts were 2.8-, 3.7-, and 3.0-fold more sensitive to bleomycin, neocarzinostatin, and mechlorethamine, respectively, than wild-type fibroblasts derived from C.B17-+/+ fetuses. These findings indicate that the scid mutation in mice causes defects in repairing both double-strand DNA breaks and DNA cross-links.

Foamy cells in non-nervous tissues from a female Siamese cat with sphingomyelinosis were examined by lectin histochemistry. Many foamy cells, so-called Niemann-Pick cells, were found in the non-nervous tissues, such as liver, spleen, lung, kidney, adrenal gland, lymph node and tonsil. These cells were positive for Concanavalia ensiformis agglutinin, Ricinus communis agglutinin-I and wheat germ agglutinin. Storage materials in the foamy cells were different from those in affected cells of the nervous tissues. This study suggests that lectin histochemistry might be helpful in the diagnosis of sphingomyelinosis.

The effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate (PDD) on VIP/PHM-27 gene expression in human neuroblastoma cells in culture were investigated. Bt2cAMP and phorbol esters increased the VIP/PHM-27 mRNA level by about 9- and 4-fold, respectively. In the presence of both Bt2cAMP and phorbol esters, the VIP/PHM-27 mRNA level increased by about 36-fold. The intracellular cAMP level was essentially unaffected by phorbol esters. The VIP/PHM-27 gene dosage was unchanged by Bt2cAMP and phorbol esters. The results suggest that cAMP and phorbol esters synergistically induce the VIP/PHM-27 gene expression through independent pathways.

A convenient and precise method for the separation and determination of coenzyme Q (CoQ)-related compounds (CoQ homologues, plastoquinone-9, ubichromenol-9, etc.) was developed using high-performance liquid chromatography (HPLC). All compounds tested were separated using a reverse-phase column with a suitable mobile phase and detected at a wavelength of 275 nm. CoQ extracts in plasma and erythrocytes were purified by thin-layer chromatography prior to HPLC analysis, but such purification was not necessary when determining CoQ in urine and tissues. Hydroquinone forms of CoQ existing in animal tissues were oxidized to the corresponding quinone forms with potassium hexacyanoferrate(III). This HPLC method was applied satisfactorily to the determination of the contents of CoQ homologues in human and animal samples. CoQ10 was the only homologue detected in human samples, and CoQ8, CoQ9 and CoQ10 were native homologues of CoQ in rat tissues. Ubichromenol-9 and plastoquinone-9 were not detected in these samples.