[Effect of peroral doxifluridine plus hepatic arterial infusion for synchronous liver metastasis of colorectal cancer--correlation with the expression of thymidine phosphorylase and dihydropyrimidine dehydrogenase in primary colorectal cancer lesions].

We investigated whether the efficacy of peroral doxifluridine and hepatic arterial 5-FU infusion on synchronous liver metastasis of colorectal cancer could be predicted based on the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in the primary colorectal lesions. Ten patients with synchronous liver metastasis of colorectal cancer were given doxifluridine (600-800 mg/body/day) orally and 5-FU (500 mg/body, once or twice a week) through the hepatic artery following resection of the primary lesions between June 1996 and July 2001. The levels of TP and DPD in the primary lesions were determined by an enzyme-linked immunosorbent assay. The level of TP, DPD, and the ratio of TP/DPD in patients with partial response (n = 4) were 89.8 +/- 30.0 U/mg protein, 23.5 +/- 25.7 U/mg protein, and 3.8 +/- 1.4, respectively, while those in patients with no response or progressive disease (n = 6) were 41.8 +/- 9.7 U/mg protein, 25.8 +/- 15.8 U/mg protein, and 2.2 +/- 1.6, showing significant difference (p < 0.01) in the level of TP between the groups. These results indicate that determining the level of TS in primary colorectal lesions may be useful for predicting the efficacy of this regimen for patients with synchronous liver metastasis of colorectal cancer.

Liver metastasis following pneumoperitoneum with different gases in a mouse model.

BACKGROUND: The validity of using CO2 in laparoscopic tumor surgery has not yet been established. To address this question, we investigated the growth of liver metastases following insufflation with different gases in a mouse laparoscopy model. METHODS: Male BALB/C mice inoculated intraportally with colon 26 cells were randomized to undergo pneumoperitoneum with CO2 (n = 16), helium (n = 16), argon (n = 16), or air (n = 17), or to act as controls without insufflation (n = 17). RESULTS: The growth of cancer nodules on the liver 14 days after surgery was greater in mice following insufflation with CO2 (p < 0.01), helium (p < 0.01), argon (p = 0.01), and air (p = 0.07) than in control mice. No significant differences were found between the four insufflation groups in the growth of liver metastases. CONCLUSION: These results suggest that insufflation plays an important role in the development of liver metastases but that the choice of gas may not affect their growth.

[Combination chemotherapy with irinotecan hydrochloride plus carboplatin for patients with advanced or recurrent colorectal cancer--a pilot study].

A pilot study was performed to evaluate the feasibility and efficacy of irinotecan hydrochloride (CPT-11) plus carboplatin (CBDCA) for treatment of advanced or recurrent colorectal cancer. Fifteen patients with colorectal cancer (nonresectable, 1; noncurative resection, 5; recurrent disease, 9) were treated with CPT-11 (40-50 mg/m2) plus CBDCA (70-100 mg/m2) once a week for 2-3 weeks followed by a one-week rest. This treatment was repeated until disease progression or severe toxic effects were found. The total dose of CPT-11 ranged from 135 to 1,214 (median, 467) mg/m2 and that of CBDCA ranged from 267 to 2,022 (median, 933) mg/m2. Adverse effects included nausea (grade 2) in 2 (13.3%) diarrhea (grade 2) in 2 (13.3%), leukopenia (grade 3) in 2 (13.3%), thrombocytopenia (grade 1) in one (6.7%), and hair falling (grade 3) in one (6.7%). The response rate of 14 evaluable patients was 14.3% (CR, 1; PR,1; NC,7; PD,5). The median survival time of all patients was 405 days from the start of chemotherapy. The survival time of patients with CR, PR, and NC (n = 9) tended to be longer than that of those with PD (n = 5) (p = 0.06). The median time to disease progression was 105 days. These results suggest that this combination chemotherapy is feasible and effective in the treatment of advanced or recurrent colorectal cancer.

The influence of different insufflation pressures during carbon dioxide pneumoperitoneum on the development of pulmonary metastasis in a mouse model.

BACKGROUND: The effects of different insufflation pressures on the development of pulmonary metastasis was investigated in a mouse laparoscopy model. METHODS: BALB/C mice intravenously inoculated with colon 26 cells were randomized to one of five treatment groups (10 mice per group): pneumoperitoneum at different pressures of 5, 10 or 15 mmHg; full laparotomy for 60 min; or anesthesia control. Cancer nodules on the lung surface 19 days postoperatively were compared between groups. RESULTS: (a) As compared with the control group, pneumoperitoneum at 10 and 15 mmHg and laparotomy enhanced the growth of pulmonary metastases (p < 0.01). (b) The growth of metastases also was greater in laparotomy group mice than in mice undergoing pneumoperitoneum at 5 and 10 mmHg (p < 0.05). CONCLUSIONS: These results suggest that the effects of different insufflation pressures on the growth of pulmonary metastases are not identical, and that pneumoperitoneum with high pressure may promote pulmonary metastases similar to those with laparotomy.

[Clinical trial of prophylactic hepatic arterial chemotherapy for liver metastases in patients with Dukes' C colorectal cancer].

Preliminary results of a prospective non-randomized trial of prophylactic hepatic arterial chemotherapy for liver metastases from colorectal cancer are presented. Twenty-two colorectal cancer patients (infusion group) in Dukes' C stage were given hepatic arterial infusion of 5-FU (500 mg/body for 1 hr per week, repeated 50 times) and peroral UFT-E (2.0 g/body, daily). Informed consent was obtained from all patients. Adverse effects and postoperative recurrence in the infusion group were compared with those of patients with UFT-E alone (control group). Complications related to hepatic arterial infusion in the infusion group were also demonstrated. There was no adverse effect in the control group, while diarrhea (grade 1) developed in one patient (5%) and pigmentation in five (24%) in the infusion group. Complications related to infusion were found in five patients (5%). Three patients in the infusion group presented with metachronous hepatic lesions, two of which were resected successfully. In the control group, one patient died of marked hepatic metastases, and one developed ovarian metastasis with lymph node involvement. Our regimen appears hematologically safe, however, the high frequency of pigmentation should be kept in mind. The oncological benefit of this chemotherapy would be clarified by a larger series of cases and longer follow-up.

[Effect of beta-glycyrrhizinate on eye mucosa absorption of drugs].

Dipotassium beta-glycyrrhizinate (GK2) is a saponin originated from "kanzo" plant. We studied such two functions of GK2 as a drug carrier and a penetration enhancer on the eye mucosa. The interactions of GK2 with various drugs were investigated using the 1-octanol/water partition method. GK2 increased the partition of cationic drugs, i.e., antihistamines (chlorpheniramine maleate (CM), diphenhydramine hydrochloride (DH)), as well as decongestants (naphazoline hydrochloride (NA), tetrahydrozoline hydrochloride (TH)). The carrier effect of GK2 is remarkable at its concentration above critical miceller concentration (CMC) and, especially at its pH value of between 4 to 5, resulting that transfers of drugs increased 2 to 10 times as compared to drugs without GK2. In vivo experiments were carried out using rats and rabbits. The effect of GK2 on the inhibition efficiency by CM was evaluated using experimental conjunctivitis formed by injection of histamine on rat upper eyelid. The Inhibition efficiency of edema by CM with GK2 was 4 times stronger than that without GK2. Cornea permeability of TH increased 1.8 times by the addition of GK2. These results indicated that GK2 is applicable as a carrier of cationic drugs on the eye mucosa and cornea.

Laparoscopic gastropexy for acute gastric volvulus: a case report.

A 7-year-old girl presented with an acute gastric volvulus that was reduced with a nasogastric catheter. An anterior gastropexy was undertaken laparoscopically. The gastrocolic omentum was deficient along most of the greater curvature, which had allowed organoaxial volvulus. Two years later, gastric volvulus has not recurred. Laparoscopy is an acceptable approach for the evaluation and treatment of children with acute gastric volvulus.

Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties.

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.

Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal influenza vaccine.

The effectiveness and safety of known mutants of Escherichia coli heat-labile enterotoxin (LT) as an adjuvant for nasal influenza vaccine were examined. Six mutants, called LT7K (Arg to Lys), LT61F (Ser to Phe), LT112K (Glu to Lys), LT118E (Gly to Glu), LT146E (Arg to Glu) and LT192G (Arg to Gly) were constructed by the replacement of one amino acid at one position of the A1 subunit to another using site-directed mutagenesis. All mutants were confirmed to be less toxic than wild-type LT when analyzed using Y-1 adrenal cells in vitro. When influenza vaccine was administered intranasally with LT7K and LT192G, BALB/c mice developed high levels of serum and local antibodies to the HA molecules. The adjuvant activity of these mutant LTs corresponded to that of wild-type LT when 1 microgram of these mutant LTs (or wild-type LT) was coadministered with the vaccine. From the point of view of safety, LT7K was considered to be the most potent mucosal adjuvant and was examined in more detail. The adjuvant activity of the mutant was lowered more rapidly with a decrease in dose than was that of wild-type LT. The low level of adjuvant of a relatively small amount of LT7K was heightened by adding LTB to the mutant LT. These results suggest that LT7K supplemented with LTB can be used as a less toxic, effective adjuvant for nasal influenza vaccine.

The beta APP717 Alzheimer mutation increases the percentage of plasma amyloid-beta protein ending at A beta42(43).

We measured plasma levels of amyloid beta protein (A beta) ending at positions 40 (A beta40) and 42(43) [A beta42(43)] in six carriers of beta APP717 (Val to Ile) mutation linked to familial Alzheimer's disease (FAD) as well as in patients with sporadic AD (sAD) and controls. The percentage and the level of A beta42(43) were significantly higher in carriers of beta APP717 mutation relative to sAD, whereas A beta40 levels were decreased. In contrast, A beta levels and ratios were at similar levels in sAD, regardless of the stage of the disease, compared with non-AD neurologic disease controls and nondemented control individuals. These results suggest that the reported increase in the percentage of A beta42(43) secretion in transfected cells with beta APP717 mutant genes actually takes place in the bodies of carriers of beta APP717 mutation, and that plasma A beta could be used as an indicator of the alterations of beta APP/A beta metabolism in subtypes of AD.

Fractionation of amyloid beta-protein (A beta) in Alzheimer's disease and Down's syndrome brains. Presence of membrane-bound A beta.

To investigate the early stage of beta-amyloidogenesis in Alzheimer's disease (AD), we performed sucrose density gradient fractionation of amyloid beta-protein (A beta) deposited in cerebral cortices from AD and Down's syndrome patients and normal aged individuals. Each fraction was subjected to Western blotting with monoclonal antibodies BA27 and BC05, which specifically recognize A beta 40 and A beta 42, respectively. The samples from brains with a large number of diffuse plaques showed strong BC05 immunoreactivity at the 1.0/1.2 M (F1) and 1.2/1.5 M (F2) interfaces. In contrast, in brains with advanced AD pathology, most of the BC05 immunoreactivity was recovered at the 1.5/2.0 M (F3) interface. In all cases, the level of BA27 immunoreactivity was negligible. Although F1A beta was determined to be A beta 1-42, it was only weakly reactive with BAN50 (monoclonal antibody raised against A beta 1-16). Delipidation of F1A beta restored full BAN50 immunoreactivity, indicating that F1A beta is bound to membrane. The present results suggest that diffuse plaques are associated with this membrane-bound A beta and thus that this novel A beta species is an initially deposited one.

G protein-mediated neuronal DNA fragmentation induced by familial Alzheimer's disease-associated mutants of APP.

Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation required the cytoplasmic domain of the mutants and appeared to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins).

Association of A beta 40-positive senile plaques with microglial cells in the brains of patients with Alzheimer's disease and in non-demented aged individuals.

To gain insight into the role of microglia in the formation of senile plaques (SP), especially in the generation of the two major molecular species of amyloid beta protein (A beta) with different carboxyl (C)-termini, A beta 40 and A beta 42(43), we conducted double immunolabelling studies on tissue sections from the brains of Alzheimer's disease (AD) and non-demented aged individuals using antibodies to the C-termini of A beta and ferritin, a marker for microglia. All SP were A beta 42(43)-positive in AD as well as in non-demented individuals, only a proportion of which were A beta 40-positive. Both in AD and in non-demented individuals, approximately 2/3 of the A beta 40-positive SP were typical SP with amyloid cores, these being almost invariably associated with microglia. A beta 40-positive, uncored SP were also frequently associated with microglia (mean, 74%), whereas only 24% of A beta 40-negative, uncored SP contained microglia. These results suggest that microglia may play a role in the maturation of SP, especially in the generation of A beta 40.

Amyloid beta protein deposition in normal aging has the same characteristics as that in Alzheimer's disease. Predominance of A beta 42(43) and association of A beta 40 with cored plaques.

Two distinct species of amyloid beta protein (A beta) with different C-termini, A beta 42(43) and A beta 40, are deposited in senile plaques (SP) of Alzheimer's disease (AD), with the former being far predominant. To investigate whether A beta 42(43) also predominates over A beta 40 in normal aging, we examined by immunocytochemistry the C-termini of A beta in SP in the brains of non-demented aged individuals and compared the results with those in AD. Virtually all SP were A beta 42(43)-positive; of these 12% in non-demented aged individuals and 25% in AD patients (mean of three areas examined) were also A beta 40-positive. In both the AD and non-demented groups, 2/3 of the A beta 40-positive SP were typical cored SP. These results indicate that A beta 42(43) is the predominant species deposited in SP in normal aging, and there is no qualitative difference in terms of the C-terminus of A beta in the parenchymal amyloid deposition between normal aging and AD.

GM1 ganglioside-bound amyloid beta-protein (A beta): a possible form of preamyloid in Alzheimer's disease.

The earliest event so far known that occurs in the brain affected with Alzheimer's disease (AD) is the deposition and fibril formation of amyloid beta-protein (A beta). A beta is cleaved from a glycosylated membrane protein, called beta-amyloid protein precursor, and normally secreted into the extracellular space. Here we report on the presence of membrane-bound A beta that tightly binds GM1 ganglioside. This suggests that this novel A beta species, rather than secreted A beta, may act as a 'seed' for amyloid and further that intracellular abnormalities in the membrane recycling already exist at the stage of amyloidogenesis.

Amyloid beta-proteins 1-40 and 1-42(43) in the soluble fraction of extra- and intracranial blood vessels.

To investigate the process of amyloid beta-protein (A beta) accumulation in cerebral amyloid angiopathy (CAA), the levels of A beta were determined in the soluble fraction of extra- and intracranial blood vessels and leptomeninges obtained at autopsy. Two enzyme immunoassays were employed that are known to sensitively and specifically quantify two A beta species, A beta 1-40 and 1-42(43). A beta was detectable in the intracranial blood vessels and leptomeninges with the latter containing the highest levels, while it was undetectable in the extracranial blood vessels. Thus the levels of soluble A beta correlated well with the predilection sites for CAA. Among individuals aged 20 to 90, the A beta levels in the leptomeninges increased sharply in those aged 50 to 70 and thereafter tended to decline. However, only slight degrees of CAA were detected by immunocytochemistry, even when those leptomeninges contained high levels of A beta comparable with those in Alzheimer's disease. The level of A beta 1-42 was almost always severalfold that of A beta 1-40 in the soluble fraction of leptomeninges. This is in good agreement with the immunocytochemical result showing the presence of A beta 40-negative, A beta 42(43)-positive meningeal vessels. These results indicate that A beta 1-42 is the initially deposited species in CAA and that the disruption of A beta homeostasis precedes A beta deposition in the meningeal vessels.

Long amyloid beta-protein secreted from wild-type human neuroblastoma IMR-32 cells.

The 39- to 43-amino acid amyloid beta-protein (A beta) is deposited as amyloid in Alzheimer's disease. Recent studies have suggested that short A beta (A beta 39 or A beta 40) and long A beta (A beta 42 or A beta 43) play different roles in Alzheimer-type pathology. However, little attempt has been made to investigate the cellular mechanisms underlying the generation of short and long A beta individually. In the present report, we first measured the amount of short and long A beta that are secreted from wild-type human and rodent cells with neuron- or glia-like properties using highly sensitive sandwich-ELISAs that discriminate long A beta from short A beta. The results showed that long A beta secreted by all cells constitutes approximately 10% of the total A beta. To identify the molecular species of long A beta, we next isolated the A beta species secreted from human neuroblastoma IMR-32 cells by affinity chromatography, gel-filtration HPLC, and reverse-phase HPLC. Mass spectrometric analysis demonstrated unequivocally that IMR-32 cells produce A beta 1-42 together with A beta 1-37, A beta 1-38, A beta 1-39, and most predominantly, A beta 1-40. Finally, to investigate the cellular mechanisms that generate A beta 1-42, we studied the effects of brefeldin A and monensin on the production of A beta 1-40 and A beta 1-42 in IMR-32 cells. These reagents reduced the production of both A beta 1-40 and A beta 1-42 simultaneously in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

The extent of amyloid deposition in brain in patients with Down's syndrome does not depend upon the apolipoprotein E genotype.

The extent of deposition of amyloid beta protein (A beta) was investigated in 20 elderly patients with Down's syndrome, using the end-specific monoclonal antibodies BC05 and BA27 to detect the presence of A beta 42(43) and A beta 40 (respectively), and related to apolipoprotein E (ApoE) genotype. No significant differences in the amount of A beta deposited in the brain, either as A beta 42(43) or A beta 40, were noted in patients possessing an ApoE E4 allele, compared to those without. Patients with an ApoE E4 allele in general died at an earlier age than those with only ApoE E3 alleles, the latter in turn being outlived by those with an ApoE E2 allele. In Down's syndrome therefore, ApoE may influence the timing of onset, or the rate of progression, of disease but without affecting the type or total amount of pathology accumulated.

Amyloid beta protein 1-42/43 (A beta 1-42/43) in cerebellar diffuse plaques: enzyme-linked immunosorbent assay and immunocytochemical study.

Diffuse plaques are immature and amorphous senile plaques and believed to be in the initial phase of plaque formation. In contrast to amyloid angiopathy and the plaque core amyloid, diffuse plaques failed to be purified in preserved forms from the brain. Here, we studied the diffuse plaques in the cerebellar region of the Alzheimer's disease brain based on immunocytochemistry and ELISA using two different monoclonal antibodies specifically recognizing the carboxyl termini of A beta molecules (BA27 for A beta 1-40 and BC05 for A beta 1-42/43). We found that the amount of A beta 1-40 was in proportion to the staining degree on amyloid angiopathy by immunohistochemistry. We found that A beta 1-42/43 comprised diffuse plaques as the major component in the cerebella of AD brains. Taking these findings into consideration, diffuse plaques, the earliest pathological change in the brain with AD, are concluded to be composed mainly of A beta 1-42/43, implicating the critical importance of this kind of A beta species deposition in the pathogenesis of AD.

Amyloid beta protein (A beta) deposition: A beta 42(43) precedes A beta 40 in Down syndrome.

The chronological relationship regarding deposition of amyloid beta protein (A beta) species, A beta 40 and A beta 42(43), was investigated in 16 brains from Down syndrome patients aged 31 to 64 years. The frontal cortex was probed with two end-specific monoclonals that recognize A beta 40 or A beta 42(43). All senile plaques detected with an authentic beta monoclonal were also A beta 42(43) positive, but only a varying proportion was A beta 40 positive. In young (< or = 50 years old) brains there were many A beta 42(43)-positive, A beta 40-negative diffuse plaques, but only few A beta 40-positive senile plaques (mean, 6.3% of total number of senile plaques). The 2 youngest Down syndrome brains showed only diffuse plaques that were all A beta 42(43) positive but A beta 40 negative. Old (> 50 years old) brains contained many mature senile plaques with amyloid cores in addition to diffuse and immature plaques and the proportion of A beta 40-positive senile plaques was increased (mean, 42% of total). Cerebral amyloid angiopathy was more abundant in old Down syndrome brains and was positive for both A beta 40 and A beta 42(43). In cerebral amyloid angiopathy, A beta 40 predominated over A beta 42(43) in both staining intensity and number of positive vessels. These results indicate that (1) the A beta species initially deposited in the brain as senile plaques is A beta 42(43) and A beta 40 only appears a decade later, and (2) in cerebral amyloid angiopathy A beta 40 appears as early as A beta 42(43).