Establishment of 2 human thyroid-carcinoma cell-lines (8305c, 8505c) bearing p53 gene-mutations

New cell lines, designated 8305C and 8505C, were established from undifferentiated thyroid carcinomas of a 67 year-old-female patient and a 78-year-old-female patient, respectively. Pathologically both these primary undifferentiated carcinoma tissues contained residual well differentiated components, suggesting well differentiated to undifferentiated carcinoma progression. Cell kinetic analysis indicate that the cell population doubling time is 43 h for 8305C and 36 h for 8505C. The saturation density at confluency is 5.7 x 10(4) cells/cm2 for 8305C and 1.1 x 10(5) cells/cm2 for 8505C. To identify genetic changes that may have occurred in these two cell lines, tumor suppressor genes p53, Rb, APC and MCC were analyzed. Sequence analysis confirmed a C:G to T:A transition at the first base of p53 gene codon 273 in 8305C and a C:G to G:C transversion at the first base of p53 codon 248 in 8505C. Polymerase chain reaction-loss of heterozygosity assays confirmed allelic deletion of p53 gene from the 8505C cell line. Loss of heterozygosity of other tumor suppressor genes were not observed. Given that p53 mutations associate with undifferentiated carcinoma but not with well differentiated carcinoma during multistep carcinogenesis of the thyroid, these cell lines should prove useful for research into the role of p53 gene mutations in malignant transformation.     

Antitumor effects of oral administration of an interferon-inducing pyrimidinone,Bropirimine, on murine renal-cell carcinoma

Bropirimine [2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone] is a low-molecular-weight compound that acts as an inducer of interferon in several animal species. Experiments were designed to explore the possibility of using this drug for the treatment of renal-cell carcinoma (RCC). Euthymic BALB/c mice were inoculated with murine RCC (Renca) cells and given graded doses of Bropirimine p.o. for 5 consecutive days beginning on day 1 following tumor inoculation. These mice were killed and tumors were excised on day 21. Bropirimine significantly (P<0.01) inhibited the tumor growth at a daily dose of 1,000 or 2,000 mg/kg. No adverse effect or toxicity was noted at 1,000 mg/kg, and at 2,000 mg/kg there was only a marginal body-weight reduction without any other appreciable side effect. In addition to the inhibition of tumor growth, there was a small yet significant (P<0.05) increase in the duration of survival (in days) in the Bropirimine-treated animals. When the treatment was delayed to begin on day 6 following tumor inoculation, Bropirimine did not suppress tumor growth in euthymic mice, pointing to the importance of the timing of the treatment. In athymic nude BALB/c mice lacking T-cells or T-cell function, Bropirimine also inhibited tumor growth (P<0.01). The antitumor effect of this drug was abolished by pretreatment with anti-asialo GM1 serum, which eliminated natural killer (NK) activity in euthymic mice. In vivo treatment with Bropirimine augmented the cytotoxicity of lymphocytes isolated from the spleens or lungs of the tumor-bearing mice, which were active against Renca and YAC-1 cells in vitro. This activity was NK-cell-dependent as judged on the basis of the results of the in vitro complement-dependent cytotoxicity assay. Since Bropirimine induced interferon (IFN)-/ production, significantly (P<0.05) elevating its serum concentration, and since this drug mimics the effects of IFN-/, it seemed likely that the Bropirimine-induced NK cell augmentation we found was mediated by IFN-/. These results suggest that Bropirimine, a booster of NK activity, may have potential as an adjunct to other therapeutic modalities in the treatment of human RCC.     

Heart rate variability in response to psychological test in hand-arm vibration syndrome patients assessed by frequency domain analysis.

To investigate heart rate variability in response to psychological tests (Japanese version of Stroop color word test and mirror drawing test) in 29 hand-arm vibration syndrome (HAVS) patients, 16 of them with vibration-induced white finger (VWF) and 13 without VWF, and 10 healthy controls of similar age, heart rate variability during spontaneous and deep (6 cycles a minute) breathing in supine position before and after exposure to the psychological tests was examined calculating frequency domain components such as low frequency (LF) power-index of both the sympathetic and parasympathetic activity, high frequency (HF) power-index of the parasympathetic activity and LF/HF-index of the sympathovagal balance. The group of all patients and the group without VWF indicated significant increase in LF/HF in the deep breathing measurement after exposure to the psychological tests. The result suggests that the sympathetic tone in the sympathovagal balance predominated in the HAVS patients which means that they had larger sensitivity of the sympathetic nervous system to the psychological tests.     

Genetic background determines the nature of immune responses and experimental immune-mediated blepharoconjunctivitis (EC)

PURPOSE: Experimental immune-mediated blepharoconjunctivitis (EC) was induced in Lewis rats by immunization with ovalbumin (OVA) in complete Freund's adjuvant (CFA) or aluminum hydroxide [Al(OH)3]. To investigate the affect of genetic factors on the susceptibility of EC, we tested different strains of rats for the development of EC. METHODS: Lewis and Brown Norway (BN) rats were immunized once with 100 microg of OVA in CFA or Al(OH)3. Three weeks later they were challenged with OVA in eye drops; 24 hours after the challenge they were sacrificed and their eyes, blood, and lymph nodes were harvested for histological studies, measurement of OVA-specific antibodies (IgG, IgG1, IgG2a, IgE), and proliferation or cytokine assay, respectively. ELISA was used to detect OVA-specific IgG; passive cutaneous anaphylaxis was used for detecting IgE. RESULTS: EC, OVA-specific IgG, and cellular immunity were induced in Lewis rats by using either adjuvant, whereas IgE was not produced by either adjuvant. In contrast, IgE was produced in BN rats using either adjuvant, whereas cellular immunity was evoked only when CFA was used. Less cellular infiltration as well as cellular proliferation was detected in BN rats immunized with Al(OH)3. In both strains, Al(OH)3 induced a higher IgG1/IgG2a ratio than did CFA. More interferon-gamma by stimulation with OVA was noted in Lewis rats compared to BN rats, whereas interleukin-4 was detected only in BN rats. CONCLUSIONS: The severity of EC evaluated by cellular infiltration was dependent on OVA-specific cellular immunity. Genetic background is more important than adjuvants in determining the nature of EC and immunity.     

Monophosphoryl lipid A provides biphasic cardioprotection against ischaemia-reperfusion injury in rat hearts

1 We utilized a rat model of myocardial infarction to investigate whether cardioprotection by monophosphoryl lipid A (MLA) is provided in the early and late phases, as well as to determine whether this cardioprotection may be related to the activation of manganese superoxide dismutase (Mn-SOD), an intrinsic radical scavenger. 2 Pretreatment with MLA (0.5 or 1.0 mg kg-1, i.v.) 24 h prior to 20-min left coronary artery (LCA) occlusion and 48-h reperfusion significantly decreased the incidence of ventricular fibrillation (VF) during ischaemia, as well as infarct size. Pretreatment with lower concentrations of MLA, however, was ineffective. 3 When we examined the time course of MLA (0.5 mg kg-1)-induced cardioprotection, both infarct size and the incidence of VF were significantly reduced in rats pretreated with MLA 0.5 h and 24 h before occlusion. We observed no differences, however, 2 and 72 h after MLA treatment. 4 The activity of Mn-SOD paralleled the cardioprotective effects of MLA. Mn-SOD activity in the myocardium was significantly enhanced in rats pretreated with MLA (0.5 mg kg-1) 0.5 and 24 h before. Mn-SOD activity was not altered, however, in rats pretreated 2 or 72 h before. Lower MLA concentrations were not effective even 24 h after the treatment. 5 We conclude that MLA treatment induced a biphasic pattern of cardioprotection. The pattern of Mn-SOD activity suggests that this enzyme may play a major role in the acquisition of cardioprotection against ischaemia-reperfusion injury.     

Control of the dispersing process and pharmacokinetics in rats for lipid A analogue, E5531.

E5531 is a synthetic disaccharide analogue of lipid A which has a low toxicity but retains the ability to reduce production of tumour necrosis factor. This analogue has potential for use in the treatment of septic shock. An injectable formulation of E5531 would be useful, but dispersion in aqueous solution is a problem. In the present study the dispersing process for E5531 was evaluated using the pH-jump method (pH 11.0-->7.3). The size of the aggregates was decreased (reaching 20 nm) with increasing dispersing time in 0.003 M NaOH (pH 11.0). The membrane fluidity of the aggregates increased with increasing dispersing time. When prepared by the normal dilution method (pH 7.3-->7.3), the size of the aggregates remained constant at 140 nm and the membrane fluidity was smaller than that of samples prepared by the pH-jump method. This indicates that during dispersing at basic pH, the hydration proceeded in a normal manner, but then stopped, just after adjustment of the pH to 7.3. This suggests that the degree of hydration of the membrane is dependent on the dispersing time at pH 11.0. Using samples with different degrees of hydration and different membrane fluidity prepared by the pH-jump method, the pharmacokinetics and stability of the aggregates were evaluated after intravenous injection into rats. The data thus obtained confirmed that the membrane fluidity was correlated with the pharmacokinetics and stability in rat plasma. It was concluded that the pharmacokinetics of E5531 in rats can be controlled by changing the degree of hydration and membrane fluidity by means of using different dispersing times in alkaline solution (pH 11.0).     

Primary infiltrating signet ring carcinoma of the eyelids

A 61-year old man presented with a five-year history of a swelling initially developing in his right lower lid that progressed to involve the lateral canthal skin and eventually the upper lid and anterior orbit. He was discovered to have an infiltrating, poorly differentiated, mucin-producing carcinoma. Systemic work-up failed to disclose a visceral malignancy, and it was concluded that his tumor was primary in the lids, arising from an adnexal sweat gland. Three other reports in the literature also share almost identical clinical and pathologic features, in that all of the earlier reports dealt with middle-aged or elderly men who had diffusely indurated lids. Histopathologically, the tumor cells grow diffusely in a sclerotic stroma, and resemble the "histiocytoid" variant of metastatic breast carcinoma to the lids in women. Ultrastructural studies in our case point toward an apocrine origin, although earlier authors have favored an eccrine origin. Despite its indolent clinical course, the tumor is capable of producing regional and distant metastases on long-term follow-up. Complete local excision, possibly necessitating radical surgery, is probably the preferred method of treatment, but local radiotherapy may have a beneficial effect in retarding spread of the disease.     

The prognostic and therapeutic relevance of p27kip1 in Ewing's family tumors.

PURPOSE: Ewing's family tumors (EFTs) display the characteristic fusion gene EWS-Fli1. We have reported EWS-Fli1 may promote the cell cycle progression accompanied by the suppression of the expression of cyclin-dependent kinase inhibitor p27(kip1) in EFT cells. Here, we describe the prognostic and therapeutic relevance of p27 in EFTs. EXPERIMENTAL DESIGN: We examined tumor samples taken from 21 patients with primary EFTs for the expression of p27 protein immunohistochemically and evaluated its correlation with clinical outcome. We also investigated the usefulness of p27 as a therapeutic strategy in vitro and in vivo using p27 expression adenovirus. Finally, we examined the process of EWS-Fli1-mediated reduction of p27 expression. RESULTS: Immunohistochemical analysis showed that a low expression level of p27 protein was related to poor event-free survival in an univariate analysis and that the expression level of p27 correlated more significantly with patient survival than several clinical factors in a multivariate survival analysis. Overexpression of p27 with the adenoviral vector remarkably inhibited the cell growth in all EFT cells tested and further induced apoptosis in the wild-type p53 EFT cells. In vivo studies demonstrated a reduction in tumor growth of EFT xenograft in nude mice treated with the intratumoral injection of p27-expressing adenovirus. EWS-Fli1 did not significantly affect the p27 promoter activity and p27 mRNA levels. However, the challenge of the proteasome inhibitor caused accumulation of p27 protein in EFT cells. These data strongly suggest EWS-Fli1 might attenuate p27 protein level via activation of the proteasome-mediated degradation pathway. CONCLUSIONS: Our findings provide the first evidence of the prognostic relevance of p27 expression in EFTs. We propose p27 as a novel and powerful therapeutic factor for the molecular target therapy of EFTs.     

Higher incidence of diabetic nephropathy in type 2 than in type 1 diabetes in early-onset diabetes in Japan

BACKGROUND: Whether the type of diabetes, race, and year and age of diagnosis affect the incidence of diabetic vascular complications is unknown. That both type 1 and type 2 diabetes occur in the young Japanese population prompted us to investigate whether the type of diabetes and the year of diagnosis are related to the incidence of nephropathy. METHODS: Of the 17,256 diabetic patients who visited the outpatient clinic at our diabetes center between 1965 and 1990, 1578 (9.1%) had early-onset diabetes (diagnosed before the age of 30); of these, 620 (39%) had type 1, and 958 (61%) had type 2 diabetes. The incidence of nephropathy was analyzed in the patients according to postpubertal duration and year of diagnosis. RESULTS: The cumulative incidence of nephropathy after 30 years of postpubertal diabetes was significantly higher (P < 0.0001) in type 2 diabetic patients (44.4%, 95% CI, 37.0 to 51.8%) than in type 1 diabetic patients (20.2%, 95% CI, 14.9 to 25.8%). The incidence of nephropathy among type 1 diabetic patients has declined during the past two decades, whereas it has not among type 2 diabetic patients. The rate ratio for type 2 diabetic patients diagnosed between 1980 and 1984 relative to type 1 diabetic patients diagnosed in the same period was 2.74 (95% CI, 1. 17 to 6.41). CONCLUSIONS: The incidence of nephropathy has declined in Japanese patients with type 1 but not in those with type 2 diabetes. In young Japanese patients, because of the higher incidence of nephropathy in type 2 diabetes and the higher prevalence of type 2 than type 1 diabetes, type 2 diabetes is likely the major cause of diabetic nephropathy.