Urinary WT1-positive cells as a non-invasive biomarker of crescent formation.

OBJECTIVE: The purpose of this study was to assess the relationship between urinary WT1-positive cells (podocytes and active parietal epithelial cells) and WT1-positive cells in renal biopsy to investigate whether urinary WT1-positive cells are useful for detection of crescent formation. METHODS: Fifty-two patients with kidney disease were investigated (15 cases with crescentic lesions and 37 cases with non-crescentic lesions) for immunoenzyme staining using anti-WT1 antibody for urine cytology and renal biopsy. Numbers of WT1-positive cells in urine and renal biopsy were counted. RESULTS: There was no correlation between urinary WT1-positive cells and WT1-positive cells in renal biopsy. However, the number of urinary WT1-positive cells in patients with crescentic lesions was significantly higher than in patients with non-crescentic lesions. In addition, the best cut-off value to detect patients with crescentic lesions using urinary was 5 cells/10-mL (area under the concentration-time curve=0.735). CONCLUSIONS: The results of our study suggest urinary WT1-positive cells can be used to detect patients with crescent formation using 5 cells/10-mL cutoff value. WT1-positive glomerular podocytes and parietal epithelial cells may be shed into urine in active glomerular disease. This study, investigating the relationship between WT1-positive cells in urine and in the renal biopsy found no correlation; however, the results do suggest that, using a cutoff value of 5 cells/10 mL, WT1 positive urinary cells can be used to detect patients with crescent formation.

A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components.

Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration.

WT1 immunoenzyme staining using SurePath(™) processed urine cytology helps to detect kidney disease.

OBJECTIVES: Damage and detachment of podocytes and loss into the urine have been implicated in the progression of kidney diseases. The purpose of this study was to investigate the potential role of urine cytology based on SurePath(™) combined with immunoenzyme staining using Wilms' tumour 1 (WT1) antibody as a podocyte marker in the discrimination of normality and non-renal urinary tract disease from kidney disease. METHODS: Sixty-six patients with kidney disease, 45 patients with lower urinary tract disease and 30 healthy volunteers were examined. Urine cytology slides were prepared using the SurePath method and immunoenzyme stained with WT1 antibody, and the number of WT1-positive cells was counted. RESULTS: In kidney disease, WT1-positive cells were found in 33 (50%) of 66 samples. No WT1-positive cells were found in 45 patients with lower urinary tract disease or in 30 healthy volunteers. The positive rates for WT1 varied with disease type, but not significantly: immunoglobulin A (IgA) nephropathy, (14/23); membranous glomerulonephritis, (4/10); Henoch-Schönlein purpura nephritis, (3/5); diabetic glomerulopathy, (5/5); minor glomerular abnormality/minimal change nephrotic syndrome (0/4). CONCLUSIONS: The results suggest that WT1 immunoenzyme staining of urine cytology can be used to detect some types of kidney disease.

Exendin-4 regulates the expression of the ATP-binding cassette transporter A1 via transcriptional factor PREB in the pancreatic ß cell line.

BACKGROUND: PRL regulatory element-binding (PREB) protein is a transcription factor that regulates insulin promoter activity in the rat anterior pituitary. The PREB protein is expressed not only in the anterior pituitary but also in pancreatic ß cells. Previously, we have reported that PREB plays an important role in glucose-mediated insulin gene expression in pancreatic ß cells. The ATP-binding cassette transporter A1 (ABCA1) in pancreatic ß cells influences insulin secretion and glucose homeostasis. Exendin-4 (Ex-4), a longacting agonist of the glucagon-like peptide 1, stimulates ABCA1 expression in pancreatic ß cells. AIMS: In this study, we examined the role played by PREB in Ex-4-induced ABCA1 expression in pancreatic ß cells. MATERIAL/SUBJECTS AND METHODS: PREB mRNA and protein expression were evaluated in pancreatic ß cell line (INS-1 cells) treated with Ex-4 (10 nM). RESULTS: Ex-4 stimulated PREB protein and mRNA expression in INS-1 cells. PREB stimulated the activity of the luciferase reporter protein that was under the control of the ABCA1 promoter. Chromatin immunoprecipitation assay showed that PREB mediates its transcriptional activity by directly binding to the ABCA1 promoter region. Finally, we used small interfering RNA to inhibit PREB expression in the cells and demonstrated that the knockdown of PREB expression attenuated the effects of Ex-4 on ABCA1 expression. CONCLUSION: PREB mediates Ex-4-stimulated transcription of the ABCA1 gene in pancreatic ß cells.

Can cytological features differentiate reactive renal tubular cells from low-grade urothelial carcinoma cells?

OBJECTIVE: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low-grade urothelial carcinoma cells (LG-UCs). METHODS: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG-UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette-like arrangement, (iv) hobnail-shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann-Whitney U-test and chi-square test, with differences considered significant at P < 0.05. RESULTS: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG-UCs. In LG-UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. CONCLUSIONS: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG-UCs.

Japanese civilian and US military interaction in the evacuation of casualties from Camp Fuji.

Historically, if US soldiers at Camp Fuji become severely ill or suffer trauma, they are transported by the ground ambulance, as the doctor-led air ambulance in eastern Shizuoka has never been permitted to land at Camp Fuji. However, it is widely recognised that severely ill or traumatised patients require time-dependent medical management. It was therefore agreed to undertake a joint exercise between the US medical assets of Camp Fuji and the doctor helicopters in eastern Shizuoka prefecture in evacuating a simulated severely ill or traumatised US soldier. The aim of this article is to describe the background and rationale between this collaboration between the civilian Japanese air ambulance and the US medical assets in Camp Fuji.

Association between post-transplantation immunoglobulin A deposition and reduced allograft function.

BACKGROUND: Post-transplantation de novo and recurrent immunoglobulin A (IgA) deposition (IgAD) in the allograft is commonly observed. However, the association between post-transplantation IgAD and reduced allograft function has not been determined. We therefore investigated the association between reduced allograft function and post-transplantation IgAD using serial allograft biopsies. METHODS: IgAD was retrospectively analyzed in 45 adults who underwent kidney transplantation for chronic glomerulonephritis, including IgA nephropathy, at Kagawa University Hospital. Allograft biopsy samples were obtained from per protocol biopsies obtained 1 and 3 years after transplantation, as well as from episode biopsies. Factors contributing to post-transplantation IgAD were assessed by calculating adjusted odds ratios (AORs) using logistic regression analysis. RESULTS: Of the 45 recipients, 18 had post-transplantation allograft IgAD. The estimated glomerular filtration rates (eGFR) 1, 2, and 3 years after transplantation were lower in the recipients with than without IgAD. Urinalysis was normal in 61% of recipients with IgAD. Reduced allograft function (eGFR <40 mL/min/1.73 m(2)) 1 year after transplantation was significantly associated with post-transplantation IgAD (AOR = 34.4 [95% CI = 2.35-502], P = .01). Conversely, blood concentrations of mycophenolic acid and latent IgAD from donor kidneys were not significantly associated with post-transplantation IgAD. CONCLUSION: Reduced allograft function may be associated with post-transplantation IgAD in the allograft.

Loss of heterozygosity in actinic keratosis, squamous cell carcinoma and sun-exposed normal-appearing skin in Japanese: difference between Japanese and Caucasians.

Actinic keratosis (AK) has been considered to be a precursor of squamous cell carcinoma (SCC). However, based on epidemiological and molecular studies, it has become questionable to regard AK as a precancerous lesion. We analyzed 37 AKs and 14 sporadic SCCs using six microsatellite markers in order to elucidate if any genetic instability or loss of heterozygosity (LOH) was implicated in tumorigenesis and progression of non-melanocytic skin tumor. Microsatellite instability (MSI) was not found in any of the AKs or SCCs indicating that genetic instability has little implication in the tumorigenesis of sporadic non-melanocytic skin tumor. LOH was found in seven of 37 lesions of AK, but in only one of 14 lesions of SCC. The significantly lower frequency of LOH than that previously reported in Caucasians suggested that the molecular pathogenesis of AKs and SCCs might be different between Japanese and Caucasians. The higher frequency of LOH in AKs than in SCCs in the present study supported the previous epidemiological and molecular studies that AK was not likely to proceed to SCC. LOH was also demonstrated in histologically normal-appearing skin in three cases suggesting that genetic alteration occurs before histological change appears in the sun-exposed skin.

A case of infantile rhabdomyofibrosarcoma with immunohistochemical, electronmicroscopical, and genetic analyses.

A case of infantile rhabdomyofibrosarcoma arising on the buttocks of a 15-month-old boy is reported with histological, immunohistochemical, electronmicroscopical, and cytogenetic findings. Histological examination showed a proliferation of spindle-shaped cells in a fasciculated pattern, with occasional rounded rhabdomyoblastic cells with abundant eosinophilic cytoplasm. Immunohistochemically, the tumor cells expressed desmin and MyoD1 but were only weakly positive for myoglobin. No clear rhabdomyoblastic features were observed by electronmicroscopic examination. Chromosome analysis showed a clone of 46, XY, der(2)t(2;11)(q37;q13), different from any karyotypic abnormality in the original report of this neoplasm. Loss of heterozygosity at 11p15.5, the most frequent genetic alteration in embryonal rhabdomyosarcoma, was not detected. The low degree of striated muscle differentiation and tumor localization supported the diagnosis of infantile rhabdomyofibrosarcoma rather than spindle-cell rhabdomyosarcoma in this case. The present case has been uneventful as of 25 months after surgery. The rather long recurrence-free period, which has not been reported in previous cases, may be attributable to chemotherapy-induced rhabdoid differentiation of the tumor cells.

Hepatic subcapsular hematoma after extracorporeal shock wave lithotripsy (ESWL) for pancreatic stones.

We present a patient with complication of huge hepatic subcapsular hematoma after extracorporeal shock wave lithotripsy (ESWL) for pancreatic lithotripsy. The hematoma measured 78-110mm. Angiography showed a subcapsular hematoma, rather than a hematoma in the liver. In the arterial phase, the distal end of the small vessel showed spotty opacification similar to microaneurysma, suggesting that it was an injury caused by separation of the liver and its capsule, caused by the shock waves. The portal vein and hepatic vein were normal. After 8 weeks of conservative therapy, the hematoma was gradually absorbed and the patient was discharged. Eight months after the accident, the hematoma had decreased to 40mm in size. After 20 months, it was completely absorbed. The reported rate of renal subcapsular hematoma after ESWL for renal or ureter stones is 0.1%-0.7%. To date, however, only five cases of hepatic subcapsular hematoma after right renal stone disintegration have been reported. This is the first report of hepatic subcapsular hematoma after ESWL for pancreatic stones.

Uterine leiomyosarcoma metastasis to the skull--case report.

A 54-year-old female presented with a rare uterine leiomyosarcoma metastasis to the skull appearing as a gross mass beneath her scalp. She had no neurological or other physical symptoms on admission. Computed tomography and magnetic resonance imaging demonstrated an enhanced dumbbell-shaped mass at the mid-frontal region beneath the scalp. The tumor was totally removed with normal surrounding bone and dura. The histological diagnosis was leiomyosarcoma. Her postoperative course was uneventful, and she received adjuvant chemotherapy. However, multiple distant bone metastases developed 1 year later. Immediate and radical resection of such tumors is recommended.

[Disproportionately large communicating fourth ventricle--report of 2 cases].

A term of 'disproportionately large, communicating fourth ventricle' (DLCFV) was first proposed by in Harwood-Nash in 1980. It is somewhat different from the well known clinical entity of 'isolated or trapped fourth ventricle', because of apparent patency of aqueductal canal. Two cases of typical DLCFV encountered in our clinic were described. First patient was a 24 year old man in whom this condition developed following operations for lumber disc and second patient was 22 year old woman in whom the disease developed after subarachnoid hemorrhage. In both cases, main symptoms were attributable to hydrocephalus but three posterior fossa symptoms, nystagmus, Parinaud' sign and truncal ataxia were also characteristic. On the CT scan, the fourth ventricle was extraordinarily enlarged. Patency of the aqueductal canal was demonstrated by air study or Conray and Metrizamide ventriculography. On the other hand, occlusion was demonstrated or highly suspected in or near the foramina Magendie and Luschka. After a routine ventriculo-peritoneal shunt operation, the fourth ventricle decreased in size and the symptoms were immediately relieved. Plausible explanation for mechanism involved in occurrence of DLCFV were (1) occlusion process in or near the fourth ventricle outlets seems to be crucial in this pathologic condition. Collision of CSF pulse waves against the obstruction may yield a water hammer effect on the fourth ventricle. (2) abnormal weakness of the brain stem parenchyma around the fourth ventricle to CSF pressure may be another contributory factor.

[Disproportionately large, communicating fourth ventricle; report of 4 cases].

Although both "Isolated Fourth Ventricle" (IFV) and "Disproportionately Large, Communicating Fourth Ventricle" (DLCFV) are the clinical-radiologic entities characterized by a dilatation of the fourth ventricle, DLCFV must be separated from IFV because of its apparent patency of the aqueduct. In some Japanese literature, however, there was some confusion concerning DLCFV and so-called "reversible DLCFV" or IFV with "one way aqueduct". In this paper, comparing DLCFV with IFV, a reasonable pathogenesis of DLCFV was discussed on the basis of clinico-radiological analysis of four cases of DLCFV. Our tentative conclusion is as follows: 1) Whether there is radiologic aqueductal patency or not, the term of DLCFV should not be primarily reserved for patients who have had shunting of the lateral ventricle for previous hydrocephalus." 2) It was strongly suggested that a mechanism involved in the development of DLCFV was the formation "membranous occlusion" in/or near the foramen Magendie.

[Chronic subdural hematoma associated with arachnoid cyst--study of the mechanism of its development].

A significant number of cases of chronic subdural hematoma associated with middle fossa arachnoid cyst has been reported in literature, but sufficiently tenable explanation for co-occurrence of both lesions has not yet proposed. In this study, authors try to elucidate mechanisms involved in development of chronic subdural hematoma and arachnoid cyst in the same patient. Eighteen cases with arachnoid cyst in the middle fossa were diagnosed by CT scan during last 5 years in our institute. Among these, five patients had chronic subdural hematoma additionally to their middle fossa arachnoid cyst. Analysis of clinical, roentgenological data and operative findings in our five cases and reviewing of cases reported so far in the literature makes clear the following characteristics in this pathological condition. 1) Patients of chronic subdural hematoma associated with arachnoid cyst were obviously younger than patients with usual chronic subdural hematoma. 2) Chronic subdural hematoma developed in the same side to the associated arachnoid cyst. 3) Characteristic changes in the skull on x-ray films indicated the long lasting existence of middle fossa arachnoid cyst. On the other hand, history of cases suggested that chronic subdural hematomas had developed within recent 1-3 months. 4) Intracranial pressure tended to remain normal or slightly elevate. 5) Abnormal, small veins which run on the surface of the membranous capsule of arachnoid cyst and bridge the Sylvian fissure were not infrequently found at operation. These veins were not able to visualized on routine angiography. On the basis of these clinical and pathological characteristics, authors infer a mechanism for development of subdural hematoma associated with arachnoid cyst. The presence of middle fossa arachnoid cyst must increase a compressibility of the intracranial content, especially of the ipsilateral cerebral hemisphere and it predisposes for development of chronic subdural hematoma.(ABSTRACT TRUNCATED AT 250 WORDS)

[Application of the polymerase chain reaction (PCR) in the diagnosis of Hb S-beta(+)-thalassemia].

Isoelectric focusing of the hemolysate prepared from a two-year-old American black boy with microcytic hypochromia showed the presence of a high percentage (63.3%) of such Hb variant as Hb S, while the levels of Hb A, Hb F and Hb A2 were 20.0%, 12.7%, and 4.0%, respectively. The ratio of the non-alpha-chain to the alpha-chain of the biosynthesized globin chains was 0.49. The variant was identified as Hb S by amino acid analysis of the abnormal peptide (beta T-1) and digestion of DNA amplified by the polymerase chain reaction with enzyme Eco 81 I. This was further confirmed by DNA sequencing. DNA sequencing of a beta-gene without the beta s-mutation revealed a nucleotide change of T to C in the polyadenylation signal sequence AATAAA 3' to the beta-gene, resulting in beta(+)-thalassemia. These results are consistent with the existence of a beta s-gene and a beta(+)-thalassemia gene in trans.

[Structural analysis of abnormal hemoglobin by the polymerase chain reaction (PCR) of genomic DNA].

Determination of the primary structure of abnormal Hbs on the basis of DNA sequencing of the globin gene obtained from a carrier of abnormal Hb was performed. DNA obtained from the leukocytes of the peripheral blood was amplified by the polymerase chain reaction (PCR) using the proper amplification primer set. Amplified DNA was digested with two different restriction endonucleases and cloned to vector M 13 mp 18 or mp 19, which had been digested with the same enzymes. DNA sequencing was done by the dideoxy chain termination method using T 7 DNA polymerase, and the abnormal Hbs whose primary structure was determined were as follows: Hb Fukuoka [beta 2 His(CAC/T)----Tyr(TAT)], Hb Machida [beta 6 Glu(GAG)----Gln (CAG)], Hb Hope [beta 136 Gly(GGT)----Asp(GAT)], Hb Hiroshima [beta 146 His(CAC)----Asp(GAC)] and Hb Kodaira [beta 146 His(CAC)----Gln(CAA)]. This method for determining the primary structure of abnormal Hbs might be more effective than the ordinary method, which involves amino acid analysis and amino acid sequencing of the abnormal peptide obtained from abnormal Hb.

[Molecular analysis of an African family with sickle cell disease and alpha-thalassemia-2].

An etiological examination was performed on the DNA of a 13-year-old Zairean girl, who had some abnormalities in hematological and red cell morphological examinations and was homozygote for an abnormal Hb like HbS. DNA was amplified by the PCR method to obtain 1.7 kb size DNA containing the 5' region of the beta globin gene. The amplified DNA was digested with Eco 81 I and electrophoresis of the digest revealed the absence of its active site, which is on codons beta 5-7 (CCTGAGGAG) of the normal DNA. Sequencing of the cloned DNA by the dideoxy method confirmed that the codon beta 6 (GAG, Glu) mutated to a new codon GTG (Val) which is the beta S globin gene producing abnormal HbS. The haplotype of the chromosome having beta S gene was --+---++, which is the most common type in Zaire area. On the other hand, when the genomic DNA was digested with Bgl II or Bam HI and hybridized to an alpha probe, a fragment (16 kb/Bgl II or 10.5 kb/Bam HI) in addition to the normal ones (12.5 and 7.5 kb/Bgl II or 14 kb/Bam HI) was observed. This resulted from the deletion of 3.7 kb from the alpha gene arrangement, which led to alpha-thalassemia-2 (genotype: alpha 3.7-/alpha alpha). A family study demonstrated that her parents were heterozygote for HbS and her father had alpha-thalassemia-2.

[Use of personal computers by diplomats of anesthesiology in Japan].

Use of personal computers by diplomats of the Japanese Board of Anesthesiology working in Japanese university hospitals was investigated. Unsigned questionnaires were returned from 232 diplomats of 18 anesthesia departments. The age of responders ranged from twenties to sixties. Personal computer systems are used by 223 diplomats (96.1%), while nine (3.9%) do not use them. The computer systems used are: Apple Macintosh 77%, IBM compatible PC 21% and UNIX 2%. Although 197 diplomats have e-mail addresses, only 162 of them actually send and receive e-mails. Diplomats in fifties use e-mail most actively and those in sixties come second.