Identification and analysis of CXCR4-positive synovial sarcoma-initiating cells.

Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target.

Intravenous administration of L-arginine inhibits angiotensin-converting enzyme in humans.

The iv administration of L-arginine, a precursor of endothelium-derived relaxing factor/nitric oxide, is known to decrease blood pressure in humans by its direct vasodilatory effects. The purpose of the present study was to determine whether L-arginine infusion modifies the renin-angiotensin (Ang)-aldosterone system as well as blood pressure and renal hemodynamics. L-Arginine and saline vehicle were iv administered to 10 healthy male subjects in random order on different days. L-Arginine infusion (500 mg/kg over 30 min) decreased mean blood pressure (from 81.2 +/- 2.7 to 74.0 +/- 2.5 mm Hg; P < 0.001) and renal vascular resistance (from 0.085 +/- 0.007 to 0.074 +/- 0.006 mm Hg/mL.min; P < 0.01) and increased heart rate (from 60.3 +/- 2.7 to 69.7 +/- 2.1 beats/min; P < 0.001) and renal plasma flow (from 616.6 +/- 37.8 to 701.0 +/- 49.2 mL/min; P < 0.05). L-Arginine reduced serum Ang-converting enzyme activity (from 10.4 +/- 0.6 to 8.9 +/- 0.5 nmol/mL.min; P < 0.05) and plasma Ang-II (from 19.3 +/- 3.3 to 12.7 +/- 2.8 pg/mL; P < 0.001), but had no effect on PRA or the glomerular filtration rate. The saline vehicle did not alter any of these parameters. The iv administration of L-arginine (endothelium-derived relaxing factor/nitric oxide) may reduce the plasma Ang-II concentration by inhibiting Ang-converting enzyme. The mechanism by which L-arginine infusion decreases blood pressure can be at least in part explained by inhibition of the renin-Ang system.

Angiotensin I-converting enzyme gene polymorphism and salt sensitivity in essential hypertension.

We undertook the present study in 66 Japanese patients with essential hypertension to identify genetic factors associated with salt sensitivity. Patients were classified into salt-sensitive or salt-resistant groups on the basis of changes in their mean blood pressures from a week of a low salt diet (50 mmol/d) to a week of a high salt diet (340 mmol/d). Salt sensitivity and resistance were studied in relation to a 287-bp insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme gene detected by a polymerase chain reaction method and the haptoglobin phenotype determined by polyacrylamide gel electrophoresis. Patients with the angiotensin I-converting enzyme gene genotype II were more apt to be salt sensitive than patients with the ID and DD genotypes, although plasma renin activity was similar in each group. The frequency of the I allele in the salt-sensitive group was significantly higher than that in the salt-resistant group (chi2 = 7.4, odds ratio = 2.78). However, there was no significant relationship between haptoglobin phenotype and salt sensitivity. These data suggest that an I/D polymorphism of the angiotensin I-converting enzyme gene is a genetic factor associated with salt sensitivity of blood pressure independently of plasma renin activity in Japanese patients with essential hypertension.

Electroencephalographic findings in children with moyamoya disease.

The EEG findings in 25 children with moyamoya disease were studied. Characteristic findings such as posterior slow, centrotemporal slow (CT slow), "rebuildup" after the end of hyperventilation, and sleep spindle depression were observed. Posterior slow activity was mainly observed in the EEGs examined within a short period (mean, 10 months) after onset, CI slow activity after a longer period (mean,, 28 months), and a diffuse low-voltage pattern after these periods (mean, 56 months). Buildup after the end of hyperventilation, which we refer to as "rebuildup," was discovered in more than half of the cases.

Establishment of a new continuous clear cell sarcoma cell line. Morphological and cytogenetic characterization and detection of chimaeric EWS/ATF-1 transcripts.

Clear cell sarcoma (CCS), a rare tumour of deep soft tissues, often has a t(12; 22) (q13; q12) translocation that induces the formation of a hybrid EWS/ATF-1 gene. To investigate these alterations further, we established a new continuous cell line directly from a CCS taken from a 9-year-old girl. The cultures were characterized with respect to morphological, ultrastructural, immunohistochemical and karyotypical features and were tested by reverse transcription PCR (RT-PCR) for chimaeric EWS/ATF-1 transcripts. The continuous cell line, designated KAO, is tumorigenic in nude mice, and the resultant tumours resemble the primary CCS. The tumour cells and the cultured cells have melanosomes in their cytoplasm and are immunoreactive with the melanoma-specific antibody HMB45, but do not express S-100 protein. The cultured CCS cells have the t(12; 22)(q13; q12) translocation and express the hybrid EWS/ATF-1 gene. No transcripts of the hybrid gene were detected in a malignant cutaneous melanoma tested simultaneously. Although CCS and malignant melanoma are morphologically related, the present results suggest that their geneses differ at the chromosome and molecular levels. They also indicate that chromosome analysis and detection of fusion EWS/ATF-1 transcripts may be useful adjuvant tools for the diagnosis of CCS.

Systemic magnesium deficiency disclosed by magnesium loading test in patients with essential hypertension.

The present study was designed to determine whether magnesium (Mg) deficiency is present in patients with essential hypertension. We measured the retention of an intravenously administered Mg load (0.2 mmol/kg MgSO4 over 4 h), and serum and erythrocyte Mg concentrations in 17 inpatients with essential hypertension and in 15 normotensive controls. There was no significant difference between the two groups in erythrocyte Mg concentration (normotensives vs., hypertensives: 2.0 +/- 0.5 vs. 2.1 +/- 0.4 mmol/l cells), serum Mg concentration (normotensives vs. hypertensive: 2.1 +/- 0.2 vs. 2.1 +/- 0.2 mg/dl), or in urinary Mg excretion (normotensives vs. hypertensives: 65.8 +/- 25.5 vs. 73.7 +/- 26.7 mg/day). However, Mg retention was significantly higher in hypertensives than in normotensives (normotensives vs. hypertensives: 31.8 +/- 12.1 vs. 41.9 +/- 13.3%). These results suggest that a systemic Mg deficiency, which is undectectable by serum or erythrocyte Mg determination, may exist in patients with essential hypertension.

Diagnosis of synovial sarcoma with the reverse transcriptase-polymerase chain reaction: analyses of 84 soft tissue and bone tumors.

The chimeric transcript SYT-SSX is generated as a result of reciprocal translocation t(X;18), which is the primary cytogenetic abnormality found in, and appears to be specific for, synovial sarcoma. We performed a reverse transcriptase-polymerase chain reaction (RT-PCR) for SYT-SSX transcripts in a series of 84 tumors (61 soft tissue tumors and 23 bone tumors), including a variety of histologic types, to assess its usefulness in molecular diagnosis. Ten synovial sarcomas, three tumors initially unclassified, and one malignant peripheral nerve sheath tumor contained the chimeric transcripts. A review of the original slides and additional examination showed that a diagnosis of synovial sarcoma was appropriate for these cases. Additionally, in situ hybridization with an SSX1 probe indicated that the chimeric transcripts exist not only in the cells of special components but also in cells showing a variety of histologic patterns. Therefore, RT-PCR can be considered a useful molecular biological technique that can provide objective evidence for diagnosis of synovial sarcoma. Northern blot analysis with an SSX1 probe also detected chimeric SYT-SSX transcripts in the synovial sarcoma cases. The additional smaller bands, however, were also detected in six peripheral primitive neuroectodermal tumors (pPNETs) and one embryonal rhabdomyosarcoma. In five of these pPNETs, other bands ranging in size from 2.0 to 2.2 kb were also found, and it seems possible that these bands might represent novel karyotypic aberrations and/or splicing variants of SSX.

Nitric oxide donor FK409 and 8-bromoguanosine-cyclic monophosphate attenuate cardiac contractility assessed by Emax.

FK409 decomposes and releases nitric oxide (NO) spontaneously when it is dissolved in phosphate buffer solution at 37 degrees C. With the use of this NO donor, the effect of exogenous NO on cardiac contractility was examined by assessing Emax. alpha-chloralose-anaesthetized dogs were instrumented for measurements of left ventricular (LV) pressure and volume and coronary blood flow (CBF) in the left anterior descending artery (LAD). FK409, 8-bromoguanosine-cyclic-monophosphate (8-Br-cGMP) and papaverine were infused into the LAD, and Emax was determined by transient inferior vena cava occlusion when CBF was increased and reached its peak. Neither drug affected heart rate nor LV pressure just before the measurement of Emax. FK409 increased CBF and decreased Emax in a dose-dependent manner. 8-Br-cGMP also increased CBF and decreased Emax in a dose-dependent manner. Pretreating with propranolol did not affect the effects of FK4098-Br-cGMP on CBF and Emax. Papaverine increased mean CBF but did not affect Emax. In conclusion NO attenuates cardiac contractility in vivo, while increasing CBF. This effect seems to be mediated by cyclic-guanosine monophosphate, a second messenger of NO.

[Studies on respiratory infections in primary care clinic (III). Distribution of bacteria isolated from patients with respiratory infections visiting 21 private clinics in the Tohoku District of Japan].

The bacteriology of the isolates from the throat swab and the sputum respectively of 2,539 patients with respiratory infections visiting 21 private clinics in Tohoku district of Japan during the period from January to April in 1989 was documented. Of the 2,539 patients, 1,694 had an acute upper respiratory infection, 609 had acute bronchitis, 46 had acute pneumonia, 84 had acute exacerbation of chronic respiratory infections and 106 had respiratory infections without diagnosis registered. 1887 (74.3%) strains of potential pathogens were recovered from 1507 (59.4%) of the 2539 cases. The rate of recovery of potential pathogens was very high in patients of the younger age. These patients had elevated body-temperature. There were statistically significant differences in recovery rate when classified by diagnosis, prefecture and the period of investigation. Of the 1,887 strains, 996 (52.8%) were gram-positive and 891 (47.2%) were gram-negative bacteria. The rate of recovery of gram-negative bacteria was high in patients who were less than 10 years old and more than 51 years old, in patients with pneumonia and chronic respiratory infections, and in patients with fever. Of the 1,887 strains, those which exceeded 100 were Staphylococcus aureus (481 strains), Haemophilus influenzae (340 strains), Streptococcus pneumoniae (329 strains), Streptococcus pyogenes (117 strains) and Acinetobacter spp. (100 strains). Species other than those mentioned above had less than 100 strains. In this group there were 39 strains of Branhamella catarrhalis, 32 strains of Escherichia coli, 97 strains of Klebsiella spp., 40 strains of Enterobacter spp., 25 strains of Serratia spp., 12 strains of Pseudomonas aeruginosa and 43 strains of Pseudomonas putida. There was a remarkable difference in recovery rate of each species when classified by diagnosis, age class, prefecture and the period of investigation, respectively. The above results indicated that gram-positive bacteria are more frequent than gram-negative bacteria, that enterobacteriaceae and glucose-non-fermentative gram-negative bacteria are only rarely found in primary care clinics, and that the bacteriology in primary care clinic is different from that of medical school-affiliated hospitals.

[Studies on respiratory infections in primary care clinic (IV). Antibiotic sensitivity of bacteria isolated from patients with respiratory infections visiting 21 private clinics in the Tohoku District of Japan].

We determined the MICs of ampicillin, methicillin, cefaclor, cefixime, cefteram, ofloxacin and ciprofloxacin against a total of 1,448 strains from 11 species: 464 strains of Staphylococcus aureus, 306 strains of Streptococcus pneumoniae, 114 strains of Streptococcus pyogenes, 37 strains of Branhamella catarrhalis, 329 strains of Haemophilus influenzae, 32 strains of Escherichia coli, 66 strains of Klebsiella pneumoniae, 26 strains of Enterobacter cloacae, 20 strains of Serratia marcescens, 12 strains of Pseudomonas aeruginosa and 42 strains of Acinetobacter calcoaceticus, isolated from the throat swab and the sputum of 2,539 patients with respiratory infections who visited 21 private clinics in Tohoku district of Japan during the period from January to April in 1989. Ciprofloxacin and ofloxacin were more active against S. aureus, B. catarrhalis, P. aeruginosa and A. calcoaceticus than other antibiotics. Ampicillin and cefteram were more active against S. pneumoniae and S. pyogenes than other antibiotics. New-quinolones and cephems of new-generation were active against H. influenzae, E. coli, K. pneumoniae, E. cloacae and S. marcescens. Of 30 strains of S. aureus which were resistant (MIC greater than or equal to 12.5 micrograms/ml) to ampicillin, only one strain was resistant (MIC greater than or equal to 12.5 micrograms/ml) to methicillin. Twenty strains (6.5%) of S. pneumoniae and 49 strains (14.9%) of H. influenzae were resistant (MIC greater than or equal to 1.56 micrograms/ml) to ampicillin. Of 101 strains of H. influenzae of which their beta-lactamase activity was determined by Nitrocephin-method, 27 (26.7%) were beta-lactamase-positive strains. The above results indicated that MRSA is only rarely found in primary care clinics but the incidence of ampicillin-resistant H. influenzae in primary care clinics is almost the same as that of the intensive care clinic, i.e. medical school-affiliated hospitals. Therefore caution should be exercised as regards antibiotic resistance of the causative organism even in primary care clinics.

[Myelodysplastic syndrome in a patient with familial Pelger-Huet anomaly].

This paper reports on a patients with congenital Pelger-Huet anomaly who developed myelodysplastic syndrome (MDS). A 45-year-old female was referred for investigation of pancytopenia of 6 months' duration. Hereditary Pelger-Huet anomaly was diagnosed by family study 7 years prior to admission. On admission, Hb was 6.5 g/dl, Ht 19.9%, Platelets 1.8 x 10(4)/microliters, and WBC 1,200/microliters with 2% myelocytes, 9% metamyelocytes, 14% bands, 2% segmented neutrophils, 58% lymphocytes and 5% monocytes. Most of the granulocytes were Pelger-Huet type with strikingly clumped nuclear chromatin. Bone marrow aspirate demonstrated 3.6% blasts and dysplastic changes including megaloblastoid features in erythroid series and micro-megakaryocytes compatible with refractory anemia, a subtype of MDS. The association of hereditary Pelger-Huet anomaly and MDS is discussed.

[Electroencephalography of Moyamoya disease].

Attempts were made to evaluate the EEG findings in 16 children and 12 adults with Moyamoya disease. (1) The children revealed specific findings such as hemispheric posterior slow (HP slow), centrotemporal slow (CT slow) and re-build-up after the end of hyperventilation. (2) HP slow was mainly observed in EEG examined within one year after the initial onset. In children in whom EEG was performed more than three years after the onset, chronic suppressive findings were found on EEG. (3) Buildup after the end of hyperventilation was revealed in almost all the children, which we called "Re-build-up". (4) No specific findings were verified on EEG in adult Moyamoya, beside slight abnormalities.