Inactivation of the p16 gene by hypermethylation and loss of heterozygosity in adenocarcinoma of the lung

We investigated the aberrant promoter hypermethylation of p16, p15 and p14 genes and loss of heterozygosity (LOH) at 9p21-22 in 48 cases of adenocarcinoma of the lung. The frequencies of hypermethylation of genes were as follows: p16, 25.0%; p15, 22.9%; and p14, 18.8%. The frequency of LOH at chromosome 9p21-22 was 60.9%. The frequency of two-hit inactivation of the p16 gene by hypermethylation and LOH was 21.7%. Two-hit inactivation of the p16 gene showed loss of protein expression and was significantly correlated with tumor size, tumor grade and the Ki-67 labeling index. Hypermethylation of the p16 gene was not significantly correlated with hypermethylation of the p15 and p14 genes, both of which are close to the p16 gene locus, suggesting that hypermethylation of these genes occurs selectivity. In conclusion, biallelic inactivation of the p16 gene by hypermethylation and LOH might cause loss of p16 expression and play an important role in the development of adenocarcinoma of the lung. Therefore, controlling and monitoring for hypermethylation of the p16 gene may be partially useful for treatment and early diagnosis of adenocarcinoma of the lung.     

Enhancement of S-antigen and its mRNA in the irides of uveitic patients

S-antigen (S-Ag) and its mRNA were analysed by immunohistochemistry and in situ hybridization in 32 iridectomy specimens from 29 uveitic patients and 10 non-uveitic patients. S-Ag was detected in one iris and its mRNA was detected in 12 uveitic patients. Neither S-Ag nor its mRNA was found in the controls (P < 0.003). Ten of the 12 patients who had detectable S-Ag mRNA, while only four of the 17 patients who did not, had received corticosteroids for more than 3 years (P = 0.006). We also demonstrated S-Ag and its mRNA in bovine iris by immunoprecipitation and polymerase chain reaction. These results indicate that S-Ag and its mRNA accumulate in the irides of some uveitic patients. This accumulation may be the result of local immunoregulatory factors and an effect of corticosteroid treatment, and may modulate ocular inflammation.     

No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

Epithelial Cell Rests of Malassez and OX6‐Immunopositive Cells in the Periodontal Ligament of Rat Molars: A Light and Transmission Electron Microscope Study

It has been shown that human and cat epithelial cell rests of Malassez (ERM) consist of heterogeneous cell populations. Immunohistochemical and immunoelectron microscopic analyses have verified the presence of neuroendocrine and Merkel‐like cells in both of these epithelia. During experimental orthodontic tooth movement, immunocompetent cells have also been found in the vicinity of ERM in rat periodontal ligament (PDL), but have not been characterized in normal rat PDL. The aim of this study was to investigate the presence and distribution of MHC class II antigen presenting cells by using OX6 antibody in ERM of rat molars by light and transmission electron microscopy. Immunohistochemical and immunoelectron microscopic observations of rat maxillary molars confirmed the presence of OX6‐positive cells in contact with ERM. Some immunopositive cytoplasmic processes containing vesicles interdigitated with cells of the Malassez epithelial clusters. Based on these findings it can be concluded that immunocompetent cells are localized close to Malassez epithelial clusters in normal rat PDL. Furthermore, the ultrastructural evidences indicate a possible interaction between the epithelial and immunocompent cells and suggest morphological and functional properties for ERM. Anat Rec, 291:242–253, 2008. © 2008 Wiley‐Liss, Inc.     

Analgesic action of loperamide,an opioid agonist,and its blocking action on voltage-dependent Ca2+ channels

We investigated the relationship between the antinociceptive effect of the opiate agonist loperamide at the spinal level and its inhibitory effect on calcium influx. Intrathecal administration of loperamide showed a significant antinociceptive effect in the formalin test, which was not prevented by naloxone. On the other hand, no significant effects were observed by nicardipine, an L-type specific blocker, or by BAY K8644, an L-type specific agonist, suggesting no significant role of L-type calcium channels in nociceptive signal transduction. Loperamide suppressed the calcium influx in dorsal root ganglion neurons. As the antinociceptive effect of loperamide was not affected by naloxone or other calcium channel blocking toxins, and loperamide showed a direct inhibitory effect on calcium-influx, the analgesic effect of intrathecally injected loperamide might be due to its blockade of the voltage-dependent calcium channels at the terminals of the primary afferent fibers.     

Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen (TAA) or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection (MOI). In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein (EGFP)-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 x g at 37 degrees C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity (MFI) of EGFP expression was about 150 (at 37 degrees C for 2 h). With centrifugal transduction (2000 x g at an MOI of 50 at 37 degrees C for 2 h), 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced (MFI: about 3100 or greater). Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction (MLR). The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC.     

Prevention of hypertensive crisis with ATP during anesthesia for pheochromcytoma

In the anesthetic management of five patients undergoing excision of pheochromocytoma, adenosine triphosphate (ATP) was used for the purpose of regulating systemic arterial pressure during the period of tumor manipulation. ATP was administered at doses of 0.05–0.4mg/kg/min. Systemic arterial pressure showed a significant decrease from 162 ± 17/103 ± 11mmHg before manipulation to 136 ± 21/81 ± 10mmHg during the manipulation period. The plasma catecholamine levels showed significant increases in this period. Immediately after excision, the systemic arterial pressure was maintained at normal levels (118 ± 13/75 ± 16mmHg) by fluid replacement and discontinuation of ATP administration, subsequently becoming 129 ± 19/79 ± 16mmHg. The heart rate was very stable and tachycardia did not ocurr during the manipulation period. Only one arrhythmic episode ocurred in one patient. The systemic vascular resistance index was significantly lower during the manipulation period than before it. It was therefore considered that ATP was useful as an agent for controlling arterial pressue during the anesthesia for pheochromocytoma.(Murata K, Sodeyama O, Ikeda K et al.: Prevention of hypertensive crisis with ATP during anesthesia for pheochromocytoma. J Anesth 1: 162–167, 1987)