Immunohistochemical study of transforming growth factor-beta 1, matrix metalloproteinase-2,9, tissue inhibitors of metalloproteinase-1,2, and basement membrane components at pancreatic ducts in chronic pancreatitis.

Little is known about the pathogenesis of fibrosis in chronic pancreatitis. To reach a better understanding of this problem, we investigated the immunolocalizations of type IV collagen (Col-IV) and laminin around pancreatic ducts, and those of matrix metalloproteinase-2,9 (MMP-2,9), tissue inhibitors of metalloproteinase-1,2), and transforming growth factor-beta 1 (TGF beta 1) at the ductal epithelia in chronic pancreatitis. This study included 20 surgical specimens of fibrotic pancreas from patients with chronic pancreatitis and five normal samples from autopsy cases. Immunostaining was performed by the streptavidin-biotin method after antigen retrieval. We evaluated the staining patterns and the percentage of positive cells of each antigen. In chronic pancreatitis, the immunostainings of Col-IV and laminin along the basement membrane (BM) of pancreatic ducts were disrupted in 11 (55%) of 20 and eight (40%) of 20, respectively, whereas no disruption was detected in normal pancreas. Positive immunostainings for MMP-2, MMP-9, TIMP-1, and TIMF-2 in ductal epithelia were 15 (75%) of 20, five (25%) of 20, four (20%) of 20, and 10 (50%) of 20, respectively, whereas no immunostaining was seen in normal pancreas. The staining intensity of MMP-2 in ductal epithelia was associated with the staining intensity of Col-IV around the pancreatic ducts. Also, the staining intensity of MMP-2 was progressively increased in proportion to the staining intensity of TGF beta 1. These findings suggest that TGF beta 1 induced in pancreatic duct cells also induced MMP-2 in an autocrine or paracrine manner, and that this MMP-2 decomposed Col-IV of the BM of pancreatic ducts in chronic pancreatitis.

Antiatherogenic effects of the antioxidant BO-653 in three different animal models.

Antioxidants have been proposed to have antiatherogenic potential by their inhibition of low density lipoprotein (LDL) oxidation. Here, we report an antioxidant, BO-653 (2,3-dihydro-5-hydroxy-2, 2-dipentyl-4,6-di-tert-butylbenzofuran), designed to exhibit antioxidative potency comparable to that of alpha-tocopherol, but yet possess a high degree of lipophilicity comparable to that of probucol. BO-653 exhibits a high affinity for LDL and is well distributed in aortic vessels in vivo. In atherosclerosis models of rabbits and mice, BO-653 has been shown to be able to suppress the formation of atherosclerotic lesions without untoward side effects. Specifically, there was no reduction of high density lipoprotein levels. This antioxidant provides additional evidence in support of the oxidized-LDL hypothesis, and itself is a promising candidate antioxidant for clinical use.

[Phase I study of TAT-59 (a new antiestrogen) in breast cancer. TAT-59 Study Group].

Phase I study of TAT-59 (Miproxifen), an antiestrogen developed in Japan for breast cancer, was conducted with the collaboration of 12 hospitals. A single dose of 1.25, 5, 10, 20, 40 and 80 mg, or 5 consecutive daily doses of 1.25, 5, 10, 20 and 40 mg/day, were given orally. After single dosing, no clinical adverse effects were found. Decrease of serum Na, Cl, Ca level and increase of serum LDH level were observed in one patient after a single dose of 5 mg of TAT. An increase in the serum LDH level was also observed in one patient after a single dose in the of 10 mg of TAT. An increase in the serum LDH level and total bilirubin, increase of eosinophil, K and milky serum were also observed in one patient after a single dose of 40 mg of TAT, respectively. All of these abnormal values returned to the normal level within 26 days after final administration of TAT. No adverse clinical findings nor abnormal laboratory findings were observed after consecutive administration of TAT. After postprandial single dosing, the time to reach the maximum serum concentration (Tmax) of DP-TAT, dephosphorylated metabolite of TAT, and its demethylated metabolite, DMDP, ranged from 5.0 to 7.3 hr and from 17.0 to 42.8 hr, respectively. The maximum serum concentration (Cmax) and AUC of DP and DMDP elevated in a dose-dependent manner. T1/2 of DP and DMDP ranged from 24.2 to 41.5, and from 91.9 to 214.7 hr, respectively. There were no significant differences between pharmacokinetics of TAT before and after food intake. Based on the above results, we concluded that a Phase II study should be conducted to evaluate the efficacy, safety and optimal dose of TAT.

Elevated plasma levels of matrix metalloproteinase-9 (92-kd type IV collagenase/gelatinase B) in hepatocellular carcinoma.

BACKGROUND: Matrix metalloproteinase 9 (MMP-9), a 92-kd gelatinase/type IV collagenase, has been implicated as playing an important role in cancer invasion and metastasis. A previous study showed that serum type IV collagenase activity correlated with metastasis by hepatocellular carcinoma (HCC). The aims of this study were to determine the plasma levels of immunoreactive MMP-9 in patients with HCC and to compare the levels with the clinical features including vascular invasion. PATIENTS AND METHODS: This study included 100 patients with HCC, 21 patients with chronic hepatitis (CH), 24 patients with liver cirrhosis (LC), and 138 healthy control subjects. Plasma MMP-9 levels were measured with a specific one-step sandwich enzyme immunoassay. RESULTS: Plasma MMP-9 levels in HCC (62 [33 to 130 ng/mL] median [25%, 75%], 13 to 660 ng/mL, minimum, maximum) were significantly elevated compared with those in normal controls (36 [25 to 45], range, 2.8-70 ng/mL), in CH (28 [18 to 30], 13 to 66 ng/mL) and in LC (35 [26 to 58], 16 to 86 ng/mL) (P < .0000001; P = .0000003; and P = .00205, respectively). When the cut-off level was defined as 60 ng/mL from a receiver operating characteristic curve, plasma MMP-9 concentrations had a sensitivity of 53% and a specificity of 89% for the detection of HCC from CH and LC. The levels were significantly higher in HCC patients with macroscopic portal venous invasion (79 [36 to 160], 15-660 ng/mL) than those without the invasion (44 [27 to 80], 13 to 210 ng/mL) (P = .00726). Plasma MMP-9 levels in patients with HCC were not correlated with tumor number, size, volume, or serum alpha-fetoprotein levels. CONCLUSIONS: The present data suggest that plasma MMP-9 levels can be a candidate for a novel marker for HCC. The levels appear to reflect its potential and ongoing activity of vascular invasion. A long-term follow-up of the patients will be necessary to determine whether increased plasma MMP-9 levels are predictive of more invasive and metastatic HCC.

[Characteristic findings of endoscopic retrograde pancreatography in the diagnosis of chronic pancreatitis with inflammatory mass].

Chronic pancreatitis with inflammatory mass (CPM) is difficult to differentiate from pancreatic cancer. To clarify the characteristic findings of CPM, we performed endoscopic retrograde pancreatography (ERP) in 39 patients. The pancreatogram showed smooth stenosis at the site of the mass in 26/39 cases (66.6%), irregular stenosis in 6 (15.4%), obstruction in 4 (10.3%), and no stenosis in 3 (7.7%). In the case of stenosis on ERP, the length of the stenosis correlated with the size of the mass. The pancreatogram distal to the mass showed dilatation in 26/35 cases (74.3%), 18 (51.4%) of which were simple dilatation. A follow-up study of 5 cases showed that all the stenoses had remained, and that the pancreatitis was aggravated in 2 cases. These findings suggest that CPM causes local stenoses of the pancreatic duct in many cases, and consequently may aggravate the stage of chronic pancreatitis.

Pyridinoline collagen cross-links in patients with chronic viral hepatitis and cirrhosis.

BACKGROUND/AIMS: The mature form of collagen cross-linking increases the resistance of collagen to degradative enzymes, and thus renders the protein in the fibrotic lesions extremely stable and the fibrosis virtually irreversible. It is crucial to elucidate the extent of cross-linking in fibrotic and cirrhotic livers if we are to control the subsequent removal of the excessive deposited collagen, whether by natural enzymes or induced by therapy. We aimed to quantitate pyridinoline, a mature form of the cross-linking, in normal control livers, viral fibrotic livers with various degrees of fibrosis and viral cirrhotic livers. METHODS: Needle liver biopsy samples from 75 patients with chronic viral hepatitis and 13 patients with viral liver cirrhosis, and six normal control livers were analyzed. Collagen and pyridinoline contents were determined by high-performance liquid chromatography. RESULTS: Significantly higher levels of pyridinoline cross-links per collagen molecule were found in the viral cirrhotic livers (0.60 [0.46, 0.65] pmol/pmol of collagen; median [25%, 75%]) compared with those in normal livers (0.39 [0.24, 0.43] pmol/pmol of collagen, p = 0.03491). But no differences were found in levels between cirrhosis and chronic hepatitis with various degrees of fibrosis. These data suggest that liver collagen may be susceptible to degradation to a similar degree in viral cirrhosis and in chronic viral hepatitis. CONCLUSION: The extent of the pyridinoline cross-linking of hepatic collagen does not seem to be responsible for the irreversibility of viral liver fibrosis.

The effect of interferon alpha and prednisolone on the serum concentrations of the aminoterminal domain of procollagen type III(PIIIP) and type IV (7-S collagen) in patients with chronic hepatitis B.

Since interferons are reported to inhibit collagen synthesis in vivo and in vitro, they are possible anti-fibrogenic drugs. However, little is known about the effect of interferons on connective tissue metabolism in liver disease. To investigate the effect, serum concentrations of the aminoterminal domain of procollagen type III and type IV (7-S collagen) were measured in 16 patients with chronic hepatitis B during alpha interferon treatment. The levels were also determined during prednisolone withdrawal treatment to investigate the effect on hepatic collagen turnover. In patients with chronic hepatitis B, the serum levels of both markers were significantly elevated before any treatment (procollagen type III: 1.10 +/- 0.39 U/ml, 7-S collagen: 6.69 +/- 3.71 ng/ml) compared with levels in 41 healthy volunteers (pro-collagen type III: 0.53 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.72 +/- 0.57 ng/ml, P < 0.001). Both levels were even more elevated during interferon therapy (procollagen type III: 1.36 +/- 0.58 U/ml, P < 0.05; 7-S collagen: 8.77 +/- 4.68 ng/ml, P < 0.01). On the other hand, serum procollagen type III and 7-S collagen levels were both decreased during prednisolone treatment (procollagen type III: 0.62 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.66 +/- 1.69 ng/ml, P < 0.01). These preliminary data suggest that interferon alpha enhanced, but prednisolone inhibited the turnover of procollagen type III and IV in the liver. Further study is required to interpret this observation in relation to anti-fibrotic therapy.

[Phase I study of NK 622 (toremifene citrate)].

A phase I study of NK 622 (toremifene citrate), a novel antiestrogen, was conducted in female patients with cancer. Patients received a single oral dosing or daily once oral dosing for five consecutive days. Any adverse effects were not experienced in the single dosing of 40 or 60 mg of NK 622. In the daily administration of 10, 20, 40, 60, 120, 240 and 480 mg/day, one of three patients who received 20 mg/day experienced grade 1 anorexia, three of four patients received 240 mg/day experienced adverse effects: Grade 1 leukopenia in one patient, Grade 1 general hot flush in one patient, and Grade 1 nausea, hot flush in the face and vertigo, Grade 2 anorexia, fatigue, dull headache and general hot flush in another one patient. These symptoms recovered to normal levels after treatment. Serum hormone levels were examined in postmenopausal patients, and a significant increase of the sex hormone binding globulin level was observed in the patients received 120 and 240 mg/day doses. Serum levels of NK 622 determined as free base (TOR) reached the peak levels in 2 to 4 hours after administration on the 1st and 5th day in daily treatment, while a metabolite N-demethyltoremifene (TOR-1) reached the peak level in 4 to 170 hours. Maximum serum levels and area under the concentration versus time curves of TOR and TOR-1 increased dose-dependently. These values also increased by repetition of the treatment. Half-lives of TOR and TOR-1 in serum ranged in 74.5 to 148.9 hours and 154.1 to 653.1 hours, respectively. From these results, it was concluded that safety and efficacy of NK 622 should be assessed by using 240 mg or less doses in clinical phase II studies where breast cancer patients received long term treatment with NK 622.

Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides.

Formation of fluorescence by the reaction of various amino acids with lipid hydroperoxides, i.e., linoleic acid 13-monohydroperoxide, methyl linoleate 13-monohydroperoxide and phosphatidylcholine hydroperoxide, in the presence of methemoglobin was investigated. Two types of fluorescence were produced: fluorescent dityrosine (3,3'-dityrosine) from tyrosine, and unidentified fluorophores with alpha- and epsilon-amino groups of various amino acids. While the former was stable after treatment with borohydride, the latter fluorophores were readily destroyed. The rate of dityrosine formation was rapid, and the yield of dityrosine was dependent on the concentrations of tyrosine and the lipid hydroperoxides. Butylated hydroxytoluene and tocopherol inhibited the formation of dityrosine, but did not affect the formation of fluorophores on the amino groups. Dityrosine appears to be formed by radical reaction of the lipid hydroperoxides, while the other fluorophores seem to be created by nonradical mechanisms.

The serum concentrations of the aminoterminal propeptide of procollagen type III and the hepatic content of mRNA for the alpha 1 chain of procollagen type III in carbon tetrachloride-induced rat liver fibrogenesis.

Serum concentrations of the aminoterminal propeptide of procollagen type III (PIIIP) are elevated in fibrogenic diseases of the liver, but the mechanism of elevation is not fully understood. To investigate the mechanism, we compared serum concentrations of PIIIP with total liver content of mRNA for the pro alpha 1 (III) chain, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Adult male rats received CCl4 in mineral oil twice weekly for 8 weeks and were compared with age-matched controls. Serum concentrations of PIIIP were measured by a specific radioimmunoassay; molecular sizes of PIIIP in serum were also determined. Pro alpha 1 (III) mRNA content in the liver was quantitated by RNA slot-blot hybridization and chemical measurement of total hepatic RNA content. Total collagen content of the liver was estimated by hydroxyproline measurement. All CCl4-treated animals had septal fibrosis after 4 weeks, and evidence of cirrhosis (regenerative nodules, ascites) was seen after 7 weeks of treatment. Serum concentrations of PIIIP and pro alpha 1 (III) mRNA content in the liver were correlated well until cirrhosis has established. They increased simultaneously after 3 weeks of treatment, 1 week before any elevation of hepatic hydroxyproline could be detected. After cirrhosis has established, pro alpha 1 (III) mRNA content in the liver decreased markedly, but serum PIIIP levels continued to be elevated. Hepatic hydroxyproline plateaued after 5 weeks. The molecular sizes of serum PIIIP indicate the release of intact native procollagen peptide during the development of cirrhosis. In conclusion, at least in CCl4-induced liver fibrosis in the rats, serum PIIIP levels can be used as a fibrogenic marker for the period progressing to cirrhosis. But the use of the serum PIIIP levels in cirrhosis seems to be limited by factors other than liver fibrogenesis.

Development of fluorescence and cross-links in eye lens crystallin by interaction with lipid peroxy radicals.

Senile nuclear cataract formation is associated with the accumulation of fluorescent insoluble proteins. alpha-Crystallin from bovine eye lens was treated with the lipid peroxy radicals generated by interaction of linoleic acid 13-monohydroperoxide or phosphatidylcholine hydroperoxide with methemoglobin. A blue non-tryptophan fluorescence composed of at least two kinds of fluorophores, stable and unstable on borohydride treatment, was produced. One of the stable fluorophores was identified as dityrosine by comparison of its retention times in amino acid analysis and HPLC with those of authentic dityrosine. Dityrosine was formed by the radical reaction, and less than 0.3% of the total tyrosine residues of the protein were transformed into dityrosine. The unstable fluorophores may be produced on the amino groups of alpha-crystallin by the non-radical reaction of the decomposition products of the radicals including monofunctional aldehyde species. Extensive intermolecular cross-links could not be explained only by the dityrosine content, but by the other modifications. Formation of fluorescence and intermolecular cross-links in alpha-crystalline in reaction with the lipid peroxy radicals suggested the participation of lipid peroxidation in the cataractous process.

Serum concentrations of the carboxyterminal cross-linking domain of procollagen type IV (NC1) and the aminoterminal propeptide of procollagen type III (PIIIP) in chronic liver disease.

Serum concentrations of both the carboxyterminal cross-linking domain (NC1) of procollagen type IV and the aminoterminal propeptide of procollagen type III (PIIIP) were measured by specific radioimmunoassays in 60 patients with chronic liver disease and 50 healthy controls. Compared with controls (5.3 +/- 1.3 ng/ml, mean +/- S.D.), NC1 concentrations were significantly elevated in patients with chronic active hepatitis (10.2 +/- 2.0 ng/ml) and liver cirrhosis (13.5 +/- 3.0 ng/ml), but not in chronic persistent hepatitis (6.0 +/- 0.9 ng/ml). The concentrations in patients with active liver cirrhosis were significantly higher than those in patients with inactive cirrhosis. Serum concentrations of PIIIP in controls, parients with chronic persistent hepatitis, chronic active hepatitis and cirrhosis were 5.8 (4.3-7.9), 5.3 (3.5-7.9), 17.5 (10.6-28.9), 16.7 (10.4-26.7) ng/ml, respectively (logarithmic mean and range of mean +/- S.D. after retransformation). Patients with liver cirrhosis had significantly higher concentrations of NC1 in serum than those with chronic active hepatitis, but there was no difference in serum PIIIP concentrations between the two groups. These data suggest an alteration of type IV collagen metabolism in chronic liver disease. In liver cirrhosis, the metabolism of collagen IV is apparently different from that of collagen type III; serum NC1 determinations may therefore provide additional information on chronic liver disease, particularly in patients with cirrhosis with a normal level of serum PIIIP. Further follow-up studies as well as investigations related to the basic mechanism of the elevation of these peptides in serum are needed in order to understand their clinical significance fully.

Fluorescent and cross-linked proteins formed by free radical and aldehyde species generated during lipid oxidation.

One of the mechanisms for the formation of lipofuscin-like fluorescent substances is considered to be related to lipid oxidation of tissues. Induction of lipid oxidation in tissues or cells produces cross-links and borohydride-reducible functions together with fluorescence in proteins. In order to elucidate the structures of fluorophores, cross-links and borohydride-reducible functions produced in proteins by lipid oxidation, the reactions of a lipid peroxy free radical with amino acids and proteins, and those of an aldehyde with primary amines were investigated. We demonstrated here two possible types of the reactions that produce the modified proteins. A peroxy free radical generated during lipid oxidation may attack the tyrosine residue in proteins to form the tyrosine radical which may be in turn dimerized into fluorescent and cross-linked tyrosine dimer. aldehyde species formed by degradation of the peroxy free radical may be polymerized into dimer, trimer, tetramer and so on, which may react with the amino groups of protein to produce fluorescence, cross-links and borohydride-reducible functions. Cross-links can be produced by the formation of Schiff base between the tetrameric dialdehyde and the amino groups of proteins.

Increased procollagen III production in patients with kidney disease.

Measurements of elevated procollagen III peptide (PIIIP) levels are used to monitor fibrosing activity in hepatic and various other diseases. Elevated PIIIP levels have also been reported in renal failure patients without such diseases. Therefore, the serum levels and renal clearance of PIIIP were investigated in 17 healthy volunteers and 100 patients with different types of acute (n = 15) and chronic (n = 85) kidney disease. PIIIP was measured by conventional and Fab radioimmunoassays. Median PIIIP levels in serum (18, range 5-55 ng/ml) and urine (34, range 1-110 micrograms/day) were significantly higher in kidney patients than serum (9, range 6-14 ng/ml) and urine levels (17, range 6-24 micrograms/day) in normal volunteers (p = 0.01). No significant differences (Kruskal-Wallis H test) were found, however, within the different kidney disease groups (acute, chronic/glomerulonephritis, interstitial nephritis). Median renal clearance of PIIIP-related peptides in kidney patients (1.5, range 0.5-2.4 ml/min) did not differ significantly (Wilcoxon U test) from that in normal volunteers (1.3, range 0.4-2.2 ml/min). These findings indicate that PIIIP elimination does not depend on renal function. PIIIP-related peptides in serum and urine, however, increase with renal failure irrespective of the activity or type of renal disease. This can be explained most probably by enhanced turnover of collagen type III by the affected kidney itself.

[Phase II study of peplomycin in breast cancer. A cooperative study. Clinical Study Group of Peplomycin for Breast Cancer in Japan].

A phase II study of peplomycin, an analogue of bleomycin, was carried out in 42 patients with advanced or recurrent breast cancer by a cooperative group consisting of 15 institutes throughout Japan, and the following results were obtained. Among the 42 patients, 38 were evaluable, in whom the overall response rate was 7.9% (3/38). For the various histologic types, the response rate was 33.3% (2/6) for papillotubular carcinoma and 9.1% (1/11) for medullary tubular carcinoma. The response rate was 33.3% (2/6) in patients without prior treatment and 3.1% (1/32) in those with prior treatment. Side effects of nausea, anorexia, malaise, alopecia and pyrexia occurred frequently, and a decrease in WBC and an increase in GOT were observed temporally. Pulmonary toxicity was observed in 7 patients.

[Clinical studies of BRL 28500 (clavulanic acid/ticarcillin) in the treatment of intraperitoneal infections and biliary tract infections].

Clinical studies have been conducted on BRL 28500 (a formulation containing 15 parts ticarcillin (TIPC) plus 1 part clavulanic acid (CVA]. BRL 28500 was administered at doses of 1.6 g or 3.2 g b.i.d., generally for 10 days by drip infusion to patients with intraperitoneal infections or biliary tract infections. Drug concentrations in the ascites were determined. A total of 76 cases was treated with BRL 28500. These cases included 49 intraperitoneal infections (suppurative peritonitis 29, postoperative peritonitis 20) and 18 biliary tract infections (cholecystitis 5, cholangitis 13). Nine cases were excluded from evaluation according to the committee's assessment. The clinical improvement as assessed by surgeons in charge increased with the duration of continued treatment and efficacies were assessed as 57.1% on day 5, 63.1% on day 7 and 77.8% on day 10 in intraperitoneal infections. Corresponding results in biliary tract infections were 38.9%, 40.0% and 42.9%, respectively. From these results, it is clear that the degree of improvement is related to the duration of treatment. The clinical usefulness as assessed by surgeons in charge of the study was 63.8% in intraperitoneal infections (suppurative peritonitis 75.0%, postoperative peritonitis 47.4%) and 58.8% in biliary tract infections (cholecystitis 100%, cholangitis 41.7%). The overall rate of usefulness was 62.5%. The clinical efficacy rates as assessed by the committee were 81.6% in intraperitoneal infections (suppurative peritonitis 93.1%, postoperative peritonitis 65.0%) and 66.7% in biliary tract infections (cholecystitis 100%, cholangitis 53.8%). In cases where causative organisms were isolated, the efficacies were 92.9% in suppurative peritonitis, 58.8% in postoperative peritonitis, 50.0% in cholangitis and overall, 69.2%. In cases from which TIPC-resistant organisms were isolated, the overall efficacy rate was 65.4% (suppurative peritonitis 88.9%, postoperative peritonitis 58.3% and cholangitis 40.0%). Regarding bacteriological effect as assessed by the committee, the eradication rate was 76.9% in intraperitoneal infections and 40.0% in biliary tract infections (71.0% overall). In cases from whom ticarcillin-resistant organisms were isolated the corresponding rates were 68.4% and 33.3% respectively, (63.6% overall). In 4 patients with peritonitis drug levels in the ascites were determined following administration of BRL 28500 by drip infusion. Good levels of both TIPC and CVA were detected 1 to 3.5 hours after administration.(ABSTRACT TRUNCATED AT 400 WORDS)