IMMUNOHISTOCHEMICAL DISTRIBUTION OF VASCULAR ENDOTHELIAL GROW TH FACTOR IN THE HUMAN PLACENTA ASSOCIATED HYDROPS FETALIS WITH

The purpose of this study was to determine the distribution and the expression of vascular endothelial growth factor VEGF in human placentas obtained from 22 cases of nonimmunologic hydrops fetalis NIHF , ranging from 25 to 41 weeks of gestation, as compared with those in 75 normal placentas. The terminal villi were examined immunohistochemically using rabbit anti-human VEGF immunoglobulin G. The expression of VEGF in the terminal villi was assessed by the ratio of the VEGF-positive area to the capillary area. Among the 22 placentas, 18 placentas were hydropic and appeared to be histologically immature, and the other 4 placentas, whose fetuses eventually recovered before their deliveries, were nonhydropic. In both the HF and normal placentas, syncytiotrophoblasts expressed VEGF in the villi throughout gestation. VEGF was also expressed by stromal cells in the terminal villi of normal placentas in only the first trimester, whereas its expression continued until the term of pregnancy in 12 of 18 HF placentas. The ratio of the VEGF-positive area to the capillary area was significantly higher in the terminal villi of all hydropic HF placentas than in those of the normal placentas but was entirely the same in the four nonhydropic placentas as in the normal control placentas. These results suggest that the persistent expression of VEGF by stromal cells in the hydropic villi of NIHF, in addition to the VEGF expression by syncytioptrophoblasts, seems to be a reaction to poor villous vascular development and to participate in the regulatory mechanisms of vascular function including angiogenesis and permeability.     

INDUCTION OF UNDIFFERENTIATED TUMORS BY JC VIRUS IN THE CEREBRUM OF RATS

Newborn Sprague-Dawley rats were inoculated intracranially with JC virus (Tokyo-1), a human polyomavirus, which had been isolated by N agashima et al. from the autopsied brain of a patient with progressive multifocal leukoencephalopathy in Japan. Twenty-one to 70 weeks later, 21 of 27 rats developed brain tumors in the cerebrum, but not in the cerebellum. Most of the tumor cells were of an undifferentiated neuroectodermal nature and showed nuclear palisades and pseudorosettes. In some tumor cells glial flbrillary acidic protein was positive immunohistochemically, and many glial filaments were demonstrated ultrastructurally. Neuronal differentiation was not proved. Two continuous lines of cultured tumor cells were established, and T antigen of JCV (Tokyo-1) was present in both cell lines. Glial differentiation was confirmed also in the tumors produced by subcutaneous transplantation of cultured tumor cells.     

Rhabdomyoblastic differentiation after chemotherapy in malignant triton tumor - a case-report

A case of malignant Triton tumor which consists of a malignant schwannoma accompanied by rhabdomyo-sarcomatous elements is reported. Only a small number is known in literature. The patient survived for four years without any sign of local recurrence or metastasis although an extremely poor prognosis for this neoplasm is reported. The therapy consisted of wide resection, irradiation and pre-and postoperative combination chemotherapy. The effect of multi-agent antineoplastic drugs on the differentiation of rhabdomyosarcoma cells and the relevant literature is discussed.     

Enhancement of ACNU cytotoxicity by pretreatment withO 6-methylguanine in ACNU-resistant brain tumors

Summary O ⁶-Methylguanine is a substrate of the DNA repair enzymeO ⁶-methylguanine-DNA methyltransferase, which is involved in the repair mechanism of DNA damage induced by chloroethylnitrosoureas such as 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). We tested the enhancement effect ofO ⁶-methylguanine pretreatment on ACNU cytotoxicity in ACNU-resistant brain tumors. Exposure toO ⁶-methylguanine at various times ranging from 2 to 48 hours increased the cytotoxic effects of ACNU on C6-1 cells, and this effect was highest at higher concentrations 500 and 1,000 M. Colorimetric cytotoxicity assay revealed at least a two-fold increase in ACNU cytotoxicity relative to controls withoutO ⁶-methylguanine. Intraarterial ACNU after treatment withO ⁶-methylguanine (two intravenous bolus injections of 80 and 40 mg/kg) significantly (P < 0.05 or P < 0.01) reduced the proliferation activity of transplanted C6-1 tumors for 96 hours after injection, whereas intravenous ACNU together withO ⁶-methylguanine significantly (P < 0.05) reduced C6-1 activity for only 48 hours. Thus, pretreatment withO ⁶-methylguanine prolonged the suppression effect of ACNU. The C6-1 tumors treated only with intravenous or intraarterial ACNU showed transient inhibition and rapid regrowth for 24 hours after treatment. These results indicate thatO ⁶-methylguanine increases ACNU cytotoxicity in anin vitro andin vivo brain tumor model.     

Expression and methylation status of the FHIT gene in acute myeloid leukemia and myelodysplastic syndrome.

To clarify the role of fragile histidine triad (FHIT) in hematological malignancies, we examined the methylation status and the expression level of the FHIT gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells in comparison with the methylation of the p15(INK4B) gene. The FHIT methylation was found in 13 of 94 (13.8%) AML and 22 of 40 (55.0%) MDS cases, but not in normal mononuclear cells (MNCs). Both the frequency and density of methylation increased in the advanced-stages MDS and the relapsed AML cases. Although FHIT and p15(INK4B) methylations were not correlated in MDS and AML, increased FHIT methylation at the relapse in AML was associated with p15(INK4B) methylation. The median expression level in AML was significantly higher than in normal MNCs, although the median expression level in those with methylation was significantly lower than in those without methylation. Furthermore, the methylation level at relapse was significantly higher than at diagnosis in AML. These results suggested that FHIT methylation was accumulated through the disease progression of MDS and AML, and the role of the FHIT gene as a tumor suppressor seemed different in AML and MDS.     

Increase of intestinal calcium absorption and bone mineral density by heated algal ingredient (HAI) in rats

Active absorbable calcium (AAACa) produced by adding HAI (heated algal ingredient) to oyster shell calcium (AACa) is quite efficiently absorbed from the intestine and can increase bone mineral density in elderly osteoporotic patients. HAI was produced by heating the seaweed Cystophyllum fusiforme under reduced pressure, extracting with 6N HCL, and partially neutralizing it. Butanol–ethanol extraction then yielded active HAI fraction A, corresponding to about 1% in weight. The active HAI fraction increased intestinal Ca absorption as shown by a dose-dependent increase of plasma Ca in young male parathyroidectomized rats maintained on a low-Ca diet by administration through a stomach tube with a constant dose of AACa. The action of the active fraction A to maintain bone mass was then tested in young male rats kept on a low-Ca diet for 2 weeks. Bone weight, trabecular bone density, and strength–strain index as indices of bone strength measured by peripheral computed tomography (pQCT) tended to increase when the active HAI fraction was given along with Ca. HAI increased intestinal Ca absorption and prevented the decrease of bone density in rats kept on a low-Ca diet. Received: July 17, 1999 / Accepted: Sept. 20, 1999     

Genetic and Serological Evidence for Multiple Instances of Unrecognized Transmission of Hepatitis C Virus in Hemodialysis Units

We investigated the unrecognized patient-to-patient transmission of hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1) of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA. A total of 24 HVR1 sequences, including the minor population of sequences of HCV isolates, from each patient were closely related and classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients were infected with HCV during an 18-month interval between examinations, and these HVR1 sequences fell into one of the five clusters. In another hemodialysis center, 5 of the 20 patients were HCV RNA positive, and two HVR1 sequences were found to be closely related and phylogenetically derived from the same cluster. The antibody responses of these patients to the HVR1 peptides representative of the genetic clusters revealed exactly the same clustering as that shown by phylogenetic analysis. These findings suggest that phylogenetic and serological analyses of HVR1 sensitively detect unrecognized and multiple transmission of HCV occurring within the same room in hemodialysis centers. Fingerprinting analyses using hypervariable regions of infectious agents are useful in identifying the precise route of transmission of infection.Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis. Screening and confirmatory assays for circulating antibodies to HCV became available (10, 27) after the molecular cloning of the HCV genome in 1989 (3). The second-generation enzyme immunoassay for detection of anti-HCV antibody has revealed a high prevalence of antibodies to HCV in hemodialysis (HD) patients (5). Most cases of HCV infection in HD patients are thought to be related to blood transfusions, but several reports from different parts of the world have also shown the presence of HCV infection in nontransfused HD patients as well (8, 22, 30), and the anti-HCV antibody-positive rates have been found to increase with the duration of dialysis (8, 22, 30). This suggests that nosocomial transmission of HCV occurs in HD units. Recently, patient-to-patient transmission of HCV in HD units has been demonstrated by molecular biology techniques, but the frequency of transmission was low (2, 20, 25). In these studies the sequences of HCV dominantly propagating in patients were determined and compared. Allander et al. (2) were the first to detect nucleotide sequence similarity in variable parts of the HCV genome in several HD patients. Sampietro et al. (20) and Stuyver et al. (25) found a rare HCV variant in several patients treated in the same HD unit by sequencing the relatively conserved region of the HCV genome, the 5′ untranslated region (5′-UTR), and the core region, respectively. However, no evidence of transmission had ever been demonstrated by sequence analysis of a minor population of HCV isolates.The HCV genome exhibits different variability of nucleotide sequences in different regions. There is a hypervariable region, hypervariable region 1 (HVR1), in the putative second envelope glycoprotein (E2) of the HCV genome (7). HVR1 consists of 27 amino acid residues located in the N-terminal region of E2 and is the most variable region in the HCV genome. Accordingly, it appeared that HVR1 might be useful for discriminating HCVs the same as a polymorphic marker for genetic fingerprinting and for detecting nosocomial transmission of HCV. It was thought that not only rare but also common genotypes of HCV might be found to be transmitted. Furthermore, most patients with chronic hepatitis C possess antibody against the HVR1 of their own isolates (9, 29), and anti-HVR1 antibody was also thought to be useful for demonstrating nosocomial transmission. In this study, we examined HCV transmission in HD units by performing phylogenetic analysis of HCV HVR1 sequences and testing for antibodies to HVR1 peptides. We also analyzed multiple sequences of HVR1 from each individual and investigated nosocomial transmission of HCV, including a minor population of HCV isolates transmitted in HD patients.     

Elevated plasma endothelin-1 level in streptozotocin-induced diabetic rats and responsiveness of the mesenteric arterial bed to endothelin-1

1. Both the plasma endothelin-1 (ET-1) levels and the plasma glucose levels were markedly elevated in streptozotocin (STZ)-induced diabetic rats.
2. The maximum contractile response of the mesenteric arterial bed to ET-1 was significantly reduced, and the vasodilatation induced by the ET_B-receptor agonist IRL-1620 in the mesenteric arterial bed was significantly reduced in STZ-induced diabetic rats.
3. ET-1 (10⁻⁸ M) caused a transient vasodilatation followed by a marked vasoconstriction in methoxamine-preconstricted mesenteric arterial beds. The ET-1-induced vasodilatation was significantly larger in beds from diabetic rats than in those from age-matched controls. By contrast, the ET-1-induced vasoconstriction was significantly smaller in STZ-induced diabetic rats than in the controls.
4. Both removal of the endothelium with Triton X-100 and preincubation with BQ-788 (10⁻⁶ M) (ET_B-receptor antagonist) abolished the ET-1-induced vasodilatation. Preincubation with BQ-485 (10⁻⁶ M) or BQ-123 (3×10⁻⁶) (ET_A-receptor antagonist) significantly augmented the ET-1-induced vasodilatation in control mesenteric arterial beds, but not that in beds from diabetic rats.
5. These results demonstrate that marked increases not only in plasma glucose, but also in plasma ET-1 occur in STZ-induced diabetic rats. We suggest that the decreased contractile response and the increased vasodilator response of the mesenteric arterial bed to ET-1 may both be due to desensitization of ET_A receptors, though ET_B receptors may also be desensitized. This desensitization may result from the elevation of the plasma ET-1 levels seen in STZ-induced diabetic rats.

     

Actions of novel antidiabetic thiazolidinedione,T-174, in animal models of non-insulin-dependent diabetes mellitus (NIDDM) and in cultured muscle cells

1. The antihyperglycaemic effect and the possible mechanism of action of T-174, a novel thiazolidinedione derivative, was determined in vivo and in vitro.
2. Oral administration of T-174 markedly improved hyperglycaemia, hyperinsulinaemia, hyperlipidaemia, and glucose intolerance in genetically obese and diabetic yellow KK (KK-A^y) mice (0.2–15.5 mg kg⁻¹ day⁻¹, for 7 days) and Zucker fatty rats (1.4–11.4 mg kg⁻¹ day⁻¹, for 6 days). The ED₅₀ values for the glucose lowering action of T-174 and pioglitazone, another thiazolidinedione antidiabetic, were 1.8 and 29 mg kg⁻¹ day⁻¹, respectively in KK-A^y mice; T-174 was about 16 times more potent than pioglitazone.
3. The hypoglycaemic effect of exogenous insulin in KK-A^y mice was enhanced after the administration of T-174. A hyperinsulinaemic euglycaemic clamp study in Zucker fatty rats showed an amelioration of whole-body insulin resistance by the T-174 treatment.
4. Insulin-stimulated glucose metabolism was enhanced in adipocytes from KK-A^y mice treated with T-174. The insulin receptor number of the adipocytes was increased without a change in the affinity of the receptor.
5. The hypomagnesaemia in KK-A^y mice was completely restored by T-174.
6. In cultured L6 myotubes, glucose consumption and [³H]-2-deoxy-glucose transport were enhanced by T-174 (EC₅₀; 6 and 4 μM, respectively). Combination of insulin with T-174 was additive to stimulate glucose disposal.
7. These results suggest that the antihyperglycaemic effect of T-174 was mediated by enhanced insulin action. This was associated with amelioration of the hypomagnesaemia and T-174 directly increased basal and insulin-stimulated glucose utilization by cultured muscle cells.

     

A comparative study on the rat aorta and mesenteric arterial bed of the possible role of nitric oxide in the desensitization of the vasoconstrictor response to an α1-adrenoceptor agonist

1. In thoracic aortic strips with intact endothelium, the first and second (1 h later) dose-response curves obtained with methoxamine were almost the same.
2. Methoxamine caused a dose-dependent increase in perfusion pressure in the rat isolated mesenteric arterial bed, but the second (1 h later) dose-response curve for methoxamine showed a significant attenuation of the response in comparison with the first.
3. The attenuation shown by the second dose-response curve for methoxamine was significantly reduced, but not abolished, in mesenteric arterial beds without endothelium. Incubating endothelium-intact mesenteric arterial beds with N^G-nitro-L-arginine (L-NOARG) caused a significant, but not complete, reversal of the attenuation shown in the second dose-response curve.
4. Incubating the mesenteric arterial bed with capsaicin, tetrodotoxin, indomethacin or with isotonic high k⁺ (60 mM) plus nicardipine did not affect the above attenuation seen in the second dose-response curve.
5. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) level in the effluent from the perfused mesenteric arterial bed was significantly increased after the second exposure to methoxamine. This effect was significantly smaller after removal of the endothelium or pretreatment with L-NOARG.
6. These results suggest that a desensitization to methoxamine develops rapidly in the mesenteric arterial bed, but not in the aorta, and that release of nitric oxide from the endothelium plays a major role in this desensitization.