Presynaptic effects of botulinum toxin type A on the neuronally evoked response of albino and pigmented rabbit iris sphincter and dilator muscles

PURPOSE: To investigate the effects of botulinum toxin type A(botulinum A toxin) on the autonomic and other non-adrenergic, non-cholinergic nerve terminals. METHODS: The effects of neurotoxin on twitch contractions evoked by electrical field stimulation (EFS) were studied in isolated rabbit iris sphincter and dilator muscles using isometric tension recording. RESULTS: Botulinum A toxin(150 nM) inhibited the fast cholinergic and slow substance P-ergic component of contraction evoked by EFS in the rabbit iris sphincter muscle without affecting the response to carbachol and substance P. Botulinum A toxin(150 nM) did not affect the twitch contraction evoked by EFS in the rabbit iris dilator muscle. CONCLUSION: These data indicated that botulinum A toxin may inhibit not only the acetylcholine release in the cholinergic nerve terminals, but also substance P release from the trigeminal nerve terminals of the rabbit iris sphincter muscle. However, neurotoxin has little effect on the adrenergic nerve terminals of the rabbit iris dilator muscle.     

Overexpression of cyclase-associated protein 2 in multistage hepatocarcinogenesis.

PURPOSE: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC. EXPERIMENTAL DESIGN: We measured the level of CAP2 mRNA by real-time quantitative PCR. We raised a polyclonal antibody against CAP2 and we confirmed the expression of CAP2 by immunoblotting and immunohistochemistry in HCC cell lines and HCC tissues. RESULTS: According to real-time quantitative PCR, the level of CAP2 mRNA was up-regulated in early HCC when compared with noncancerous liver tissue, and it was further up-regulated in progressed HCC. We raised a polyclonal antibody against CAP2, which showed a single 53-kDa band of strong intensity in the human HCC cell lines and HCC tissues but only a weak band in the noncancerous liver tissues in Western blot analysis. Immunohistochemical examination of CAP2 revealed its significant overexpression in early HCC when compared with noncancerous and precancerous lesions and in progressed HCC when compared with early HCC. CONCLUSION: Our findings show that CAP2 is up-regulated in HCC when compared with noncancerous and precancerous lesions. This is the first report that proves that CAP2 is up-regulated in human cancers and that this is possibly related to multistage hepatocarcinogenesis.     

Critical involvement of the phosphatidylinositol 3-kinase/Akt pathway in anchorage-independent growth and hematogeneous intrahepatic metastasis of liver cancer

In the multistep process of metastasis, "anchorage-independent growth," where cancer cells need to survive without cell-substratum interaction, is supposed to be important. In this study, we found that anchorage-independent growth analyzed using the soft agar colony formation assay correlated with hematogeneous intrahepatic metastasis of liver cancer cell lines and also Akt activation status. Two highly metastatic liver cancer cell lines showed high Akt activity and formed many colonies in soft agar, whereas three nonmetastatic cell lines showed less Akt activity and formed fewer colonies. Inhibition of Akt activation in the highly metastatic cell line Li7 by transfection with kinase-dead Akt or the phosphatidylinositol 3-kinase inhibitor, LY294002, resulted in formation of fewer colonies in soft agar than was the case with control cells. Moreover, in orthotopic implantation model, this inhibition resulted in a reduced rate of hematogeneous intrahepatic metastasis. These findings indicated that anchorage-independent growth regulated by phosphatidylinositol 3-kinase/Akt pathway plays a critical role in metastasis, and that this could be a potential therapeutic target to combat metastasis.     

FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis.

PURPOSE: Tumor-infiltrating lymphocytes represent the host immune response to cancer. CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress the immune reaction. The aim of the present study was to investigate the clinicopathologic significance and roles of Tregs and CD8+ T cells during hepatocarcinogenesis. EXPERIMENTAL DESIGN: We examined the infiltration of FOXP3+ Tregs and CD8+ T cells in the tumor stroma and nontumorous liver parenchyma using 323 hepatic nodules including precursor lesions, early hepatocellular carcinoma (HCC), and advanced HCC, along with 39 intrahepatic cholangiocarcinomas and 59 metastatic liver adenocarcinomas. We did immunohistochemical comparative studies. RESULTS: The prevalence of Tregs was significantly higher in HCC than in the nontumorous liver (P<0.001). The patient group with a high prevalence of Tregs infiltrating HCC showed a significantly lower survival rate (P=0.007). Multivariate analysis revealed that the prevalence of Tregs infiltrating HCC was an independent prognostic factor. The prevalence of Tregs increased in a stepwise manner (P<0.001) and that of CD8+ T cells decreased during the progression of hepatocarcinogenesis (P<0.001). Regardless of the presence of hepatitis virus infection or histopathologic evidence of hepatitis, the prevalence of Tregs was significantly increased in nontumorous liver bearing primary hepatic tumors. CONCLUSIONS: Tregs play a role in controlling the immune response to HCC during the progression of hepatocarcinogenesis. It has been suggested that primary hepatic cancers develop in liver that is immunosuppressed by a marked infiltration of Tregs. A high prevalence of Tregs infiltrating HCC is thought to be an unfavorable prognostic indicator.     

Postoperative management in single-lung transplantation for lymphangioleiomyomatosis

A 35-yr-old woman presented with dyspnea has been diagnosed as having lymphangioleiomyomatosis (LAM). Despite treatment with estrogen, her pulmonary function deteriorated progressively. In January 2001, a left single-lung transplantation was performed on her from a cadaveric donor. On admission to the ICU after the transplantation, arterial blood gas analysis showed a severe respiratory acidosis. A double-lumen endotracheal tube (ETT) was replaced by a single-lumen ETT for a better suctioning of secretion. Gas exchange improved after the replacement of ETT and suctioning of secretion by bronchoscopy. Five hours after the admission to the ICU, the breath sound decreased over the right thorax. The chest X-ray showed right pneumothorax, and a chest tube was inserted. The patient was weaned from mechanical ventilation gradually and extubated on the 6th ICU day. The patient was discharged from ICU to the general ward on the 9th ICU day without pneumonia and other complications. The development of pneumothorax in the recipient lung should be kept in mind during the perioperative period of lung transplantation for LAM.     

Application of bispectral index (BIS) monitoring to anesthetic management of posterior spinal fusion in a patient with Duchenne muscular dystrophy

A 12-year-old boy with Duchenne muscular dystrophy underwent posterior spinal fusion for progressive scoliosis. Preoperative evaluation was focused on respiratory function as well as cardiac function, which revealed markedly reduced respiratory reserve (FVC 0.77 l, %FVC 25.9%, FEV1.0 0.48 l, %FEV1.0 62%) and well-preserved biventricular function. A possible association between malignant hyperthermia and Duchenne muscular dystrophy has been documented. Thus anesthesia was administered without triggering agents. Propofol and fentanyl were used for induction and maintenance of anesthesia, and the patient was ventilated with O2-nitrous oxide mixture. The anesthesia machine, prepared by using a disposable circuit and fresh CO2-absorbent and disconnecting the vaporizers, was flushed with O2 at a rate of 10 l.min-1 for 20 minutes before use. A small dose of vecuronium was administered while monitoring the train-of-four ratio. The bispectral index (BIS) was utilized to optimize the depth of anesthesia so that the wake-up test could be performed promptly on surgeon's request while avoiding the intraoperative awareness. The BIS was helpful in continuously assessing the wakening process. BIS increased from 40's to 80's in 15 minutes after discontinuation of propofol and nitrous oxide during the test. The patient was kept under close observation postoperatively without any sign of malignant hyperthermia.     

Laminin 5 beta3 and gamma2 chains are frequently coexpressed in cancer cells

Laminin 5 plays an important role in cell migration during tumor invasion and tissue remodeling. However, previous studies have not clarified whether laminin 5 beta3 chain is coexpressed with laminin 5 gamma 2 chain in cancer cells. The present immunohistochemical study investigated the distribution of the laminin 5 beta3 and gamma2 chains in 20 cases of squamous cell carcinoma of the tongue and 17 cases of colorectal carcinoma. Laminin 5 beta3 and gamma2 chains were expressed in the cytoplasm of tumor cells. Tumor cells at the cancer-stromal interface and at the invasive front frequently showed immunopositivity for laminin 5 beta3 and gamma2 chains in both squamous cell carcinoma of the tongue and colorectal carcinoma. Furthermore, strong expressions of these two proteins were observed in cancer cells invading in a scattered manner. Laminin 5 beta3 expression correlated significantly with laminin 5 gamma2 expression in 20 cases of squamous cell carcinoma of the tongue (P = 0.0002) and 17 cases of colorectal carcinoma (P < 0.0001). These results suggest that in squamous cell carcinoma of the tongue and colorectal carcinoma, laminin 5 gamma2 chain and beta3 chain are both important in the invasiveness of cancer cells.     

Chronic effects of ethanol on cultured myocardial cells: ultrastructural and morphometric studies

Ultrastructural alterations of the myocardium due to chronic ethanol exposure were investigated using an in vitro system-mouse ventricular myocardial cells in a monolayer culture, which were spontaneously and synchronously contracting-by chronic exposure to 12.5, 50, and 200 mM ethanol for up to 21 days. Morphometric analyses revealed that exposure to 12.5 mM ethanol for 14 days induced an increase in the number of residual bodies, which are lysosomes containing electron-dense, amorphous materials. Some cells exposed to 50 mM ethanol for 14 days contained an accumulation of glycogen granules, increasing in inverse proportion to the mitochondrial volume. The volumetric proportion of myofibrils on day 14 decreased as the ethanol dose became lower, and was in proportion to large and giant mitochondria within the limits of three ethanol groups. Dose-dependent increases in the size and volumetric proportion of mitochondria were observed after the 14-day exposure; at a low dose (12.5 mM) mitochondria of usual size tended to increase, whereas at a high dose (200 mM) giant mitochondria increased. Coincidentally with this mitochondrial increase or gigantism, all ethanol groups showed higher beat rates than the control. Consequently, it is most likely that chronic 14-day exposures to these three ethanol doses remodel the cellular function of the in vitro myocardium in different ways; the 200-mM dose induced mitochondrial hypertrophy, an adaptive response to switch myocardial energy metabolism over to some special one; the 50-mM dose was a boundary dose; and the 12.5-mM dose mostly mimicked the chronic in vivo administration of ethanol and induced slightly degenerative alterations-increased residual bodies and lysosomes, decreased myofibrils and lowered mitochondrial respiratory function.     

Correlation of DNA hypomethylation at pericentromeric heterochromatin regions of chromosomes 16 and 1 with histological features and chromosomal abnormalities of human breast carcinomas

Changes in DNA methylation status are not only important for regulating gene expression but are also suggested to induce chromosome instability. To reveal the correlation of DNA methylation status in heterochromatin regions with tumor histology and with chromosome alterations, DNA methylation status was examined by Southern blot analysis, and numerical and structural chromosome alterations, including the formation of der(16)t(1;16)/der(1;16), were examined by fluorescence in situ hybridization at the two loci in the pericentromeric satellite 2 regions of chromosomes 16 and 1 in 39 human breast carcinomas. DNA hypomethylation at the D16Z3 and the D1Z1 loci was detected in 31% (12 of 39) and 36% (12 of 33) of carcinomas, respectively, and mostly concurred. DNA hypomethylation was more frequent in the carcinoma group of more aggressive histological types or grade 3 than in the carcinoma of less aggressive histological types or grades 1 and 2, and tended to be more frequent in carcinomas with > or =4 copies of chromosomes 16 and/or 1 than in carcinomas with < or =3 copies of any of these chromosomes. The frequency of DNA hypomethylation at the D16Z3 and the D1Z1 loci was 45% (10 of 22) and 53% (9 of 17) in carcinomas without der(16)t(1;16)/der(1;16), formation, but only 12% (2 of 17) and 19% (3 of 16) in carcinoma with der(16)t(1;16)/der(1;16), respectively (P = 0.036 and 0.070). The 16q breakage was almost equally detected between carcinoma groups with and without the DNA hypomethylation. DNA hypomethylation in the satellite 2 regions was suggested to be associated with the accumulation of a large number of numerical chromosome alterations and involved in the development of breast carcinomas of aggressive histological features. On the contrary, chromosome instability induced by mechanisms other than DNA hypomethylation in heterochromatin regions might cause the formation of der(16)t(1;16)/der(1;16) and less aggressive breast carcinomas.