Detection of mutations in the enhancer 2/core promoter region of hepatitis B virus in patients with chronic hepatitis B virus infection: comparison with mutations in precore and core regions in relation to clinical status

To investigate the meaning of the mutations in the enhancer 2/core promoter (Enh2/CP) region of hepatitis B virus (HBV) during the chronic HBV infection, mutations were examined in the Enh2/ CP region (carboxyl half of X region) and their correlation with mutations in the precore and core regions in relation to the presence of chronic liver disease. The entire nucleotide sequences of the Enh2/CP region were determined by direct sequencing of the amplified products derived from 30 cases with chronic HBV infection. The results were compared to the mutations in the precore and core regions. In the Enh2/CP region, 91 generally scattered nucleotide substitutions were detected. There were 11 substitutions in the 10 asymptomatic healthy carriers (mean, 1.1/case) and 80 in the 20 chronic liver disease patients (4.0/case). The most frequent substitutions from A to T at nucleotide 1764 and from G to A at nucleotide 1766 were seen in none of the 10 asymptomatic carriers and in 14 (70%) of the 20 chronic liver disease patients. Comparisons of mutations in the precore and core regions revealed that 14 of 16 patients with mutations in the core region had the mutations in the Enh2/CP region and/or a precore stop codon mutation. These data suggest that mutations in the Enh2/CP and precore regions may affect the expression of the core and HBeAg peptides and might be involved in the pathogenesis of chronic liver disease.     

Balloon cell nevus of the soft palate: an immunohistochemical and ultrastructural study

An ultrastructural analysis of oral balloon cell nevus of intramucosal type complemented with an immunohistochemical study was performed for the first time. The lesion was composed of large balloon cells with an admixture of small nevus cells and melanophages at the periphery. Balloon cells showed cytoplasmic accumulation of vacuoles of varying sizes and the presence of microgranular and vacuolated melanosomes were found. Residual cytoplasm contained no identifiable organelles. A spectrum of transitional forms between balloon cells and conventional nevus cells with microvacuoles was readily observed. Both cells exhibited intense immunoreactivity to multiple melanocytic markers. Ballooning phenomenon was not evident in melanophages containing a large amount of melanosome complex. It can be inferred, from the present and previous observations, that progressive vacuolization of melanosomes in nevomelanocytes may be responsible for the formation of peculiar ballooning appearance, suggesting an aberrant melanogenesis.     

Protective effects of a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia and Agaricus blazei murill against X-irradiation in B6C3F1 mice: Increased small intestinal crypt survival and prolongation of average time to animal death

Radioprotective effects of a water-soluble extracts from cultured medium of Ganoderma lucidum (Rei-shi) mycelia (designed as MAK) and Agaricus blazei (Agaricus) against the shortening of survival time or the injury of crypt by X-irradiation were investigated in male B6C3F1 mice. MAK and Agaricus at three different doses were mixed into basal diet into biscuits at 5, 2.5 and 1.25% and administered from 1 week before irradiation. MAK (5% group) significantly prolonged animal survival as compared with basal diet group (control group) after 7 Gy of X-ray irradiation at a dose rate of 2 Gy min(-1). At doses of 8, 10 and 12 Gy X-irradiation at a dose rate of 4 Gy min(-1) MAK (5% group) significantly increased crypt survival as compared to other groups. These results suggest that MAK can act as a radioprotective agent.     

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.     

Positron CT imaging using a high resolution PCT device (Positologica-I),11CO,13NH3, and18FDG in clinical evaluation of cerebrovascular diseases

Positron computed tomography (PCT) was performed in 3 normal volunteers and 21 patients with cerebrovascular diseases using a high resolution PCT device Positologica-I and three tracers¹¹CO,¹³NH₃, and¹⁸FDG. Relatively early lesions showed various accumulation patterns, and metabolism and perfusion mismatches were clearly shown by this measurement. One type of mismatch is luxury perfusion which had a slight increase of blood volume. Another type of uncoupling is misery perfusion. Remote effects of ischemic lesions also appeared on PCT with¹⁸FDG and¹³NH₃. From our clinical results, the PCT method with a high resolution device and radiopharmaceuticals such as¹¹CO,¹³NH₃, and¹⁸FDG is very useful in the assessment of cerebrovascular diseases and in defining circulatory dysfunction in man.     

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.     

In Vitro Contraction of the Canine Corpus Cavernosum Penis by Direct Perfusion with Prolactin or Growth Hormone

Purpose

It is well established that hyperprolactinemia, most typically seen in prolactinoma patients, causes hypogonadism and impotence. There seem to be a good possibility that hyperprolactinemia causes impotence, at least partially via some intrinsic property of prolactin (PRL), rather than through its suppressive effects on the hypothalamic-pituitary-gonadal testosterone dynamics. In the present investigation, we used an in vitro canine model to attempt to clarify whether direct action of PRL on the corpus cavernosum penis may lead to erectile insufficiency. Growth hormone (GH) and placental lactogen (PL), both having close structural and functional homologies to PRL, were also studied.

Materials and Methods

Isometric tension measurement with cavernous strips was performed in the presence or absence of 10 sup -5 to 10 sup -9 M. PRL, GH, or PL in the perfusion medium. The tension change induced by the test substances was normalized relative to that induced by 120 mEq KCl.

Results

Both PRL and GH produced dose-related elevations (p less than 0.01) of the cavernous tension, whereas PL and thiol-cleaved PRL in comparable doses were without effect (p greater than 0.05). When the tension rise produced by 120 mEq KCl was taken as 100 percent, the maximum contractions produced by PRL and GH were 80 percent and 110 percent. The minimum effective concentration was 10 sup -8 to 10 sup -7 M. for both PRL and GH. Pretreatment with indomethacin (10 sup -5 M.), but not tetrodotoxin (10 sup -5 M.), partially suppressed (p less than 0.05) the effects of PRL.

Conclusion

These results suggest that PRL and GH directly and specifically produced contraction of the corpus cavernosum penis, resulting in erectile insufficiency, and that the effect of PRL is partially mediated by prostaglandin.