Correlation of serum ribavirin concentration with pretreatment renal function estimates in patients with chronic hepatitis C receiving combination antiviral therapy with peginterferon and ribavirin.

Serum ribavirin concentration is an important factor in antiviral therapy in combination with peginterferon (PEG-IFN) and ribavirin for patients with chronic hepatitis C in terms of both beneficial and adverse effects. We evaluated whether the serum ribavirin concentration can be predicted on the basis of renal function estimates. Serum creatinine and cystatin C concentrations were measured at the start of treatment in a total of 148 patients with chronic hepatitis C who underwent combination PEG-IFN and ribavirin therapy. Creatinine clearance (CrCl) and total clearance of ribavirin (CL/F) were calculated on the basis of the serum creatinine level. The glomerular filtration rate was calculated with two different formulae on the basis of the serum cystatin C level. These values were compared with serum ribavirin concentrations 4 weeks after the start of therapy. The cystatin C level increased with the progression of liver fibrosis, whereas the creatinine level was constant regardless of the degree of liver fibrosis. Significant correlation was not observed between the serum ribavirin concentration and serum creatinine level, cystatin C level, or calculated renal function estimates. However, significant correlation was found between the serum ribavirin concentration and CrCl and CL/F in patients who were given ribavirin >800 mg/day. Overall, renal function estimates do not correlate with the serum ribavirin concentration in Japanese patients with chronic hepatitis C who undergo combination PEG-IFN and ribavirin therapy. Serum creatinine-based renal function estimates might be predictive for the serum ribavirin concentration only in patients with a daily ribavirin intake of 800 mg or more.

Viral eradication reduces both liver stiffness and steatosis in patients with chronic hepatitis C virus infection who received direct-acting anti-viral therapy.

BACKGROUND: Whether direct-acting anti-viral therapy can reduce liver fibrosis and steatosis in patients with chronic hepatitis C virus (HCV) infection is unclear. AIMS: To evaluate changes in liver stiffness and steatosis in patients with HCV who received direct-acting anti-viral therapy and achieved sustained virological response (SVR). METHODS: A total of 198 patients infected with HCV genotype 1 or 2 who achieved SVR after direct-acting anti-viral therapy were analysed. Liver stiffness as evaluated by magnetic resonance elastography, steatosis as evaluated by magnetic resonance imaging-determined proton density fat fraction (PDFF), insulin resistance, and laboratory data were assessed before treatment (baseline) and at 24 weeks after the end of treatment (SVR24). RESULTS: Alanine aminotransferase and homeostatic model assessment-insulin resistance levels decreased significantly from baseline to SVR24. Conversely, platelet count, which is inversely associated with liver fibrosis, increased significantly from baseline to SVR24. In patients with high triglyceride levels (≥150 mg/dL), triglyceride levels significantly decreased from baseline to SVR24 (P = 0.004). The median (interquartile range) liver stiffness values at baseline and SVR24 were 3.10 (2.70-4.18) kPa and 2.80 (2.40-3.77) kPa respectively (P < 0.001). The PDFF values at baseline and SVR 24 were 2.4 (1.7-3.4)% and 1.9 (1.3-2.8)% respectively (P < 0.001). In addition, 68% (19/28) of patients with fatty liver at baseline (PDFF ≥5.2%; n = 28) no longer had fatty liver (PDFF <5.2%) at SVR24. CONCLUSION: Viral eradication reduces both liver stiffness and steatosis in patients with chronic HCV who received direct-acting anti-viral therapy (UMIN000017020).

Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001.

The 5' upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the 1. 1-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A. xylinum and E. coli.

Effect of particles on sheep lung hemodynamics parallels depletion and recovery of intravascular macrophages.

We previously showed in newborn lambs that the pulmonary hemodynamic responses to foreign particulate matter (liposomes; Monastral blue) developed in parallel with the maturation of the pulmonary intravascular macrophage system. We now report our use of the liposome-encapsulated heavy-metal-chelating agent dichloromethylene diphosphonate to deplete the intravascular macrophages of small lambs. Functionally and by quantitative histology, we depleted the vast majority of the intravascular macrophages (71% by Monastral blue particle retention, n = 22; 77% by histology; n = 2). Depletion success increased to > 90% as we optimized the liposome-depletion regime. Recovery of the lung hemodynamic response began within 3 days. By 2 wk, the functional responses had fully recovered (n = 8), and, according to quantitative histology, the macrophage population (n = 2) had recovered 65%. Macrophage depletion in lambs is relatively inexpensive and easy to achieve. It is a safe procedure and is followed by full recovery in approximately 2 wk, provided that an aseptic technique is used to prevent bacteremia.

Intravascular macrophage depletion attenuates endotoxin lung injury in anesthetized sheep.

We recently showed that we can selectively and safely deplete most (average 85%) of the pulmonary intravascular macrophages in sheep by intravenously infusing liposomes containing dichloromethylene bisphosphonate. After a 1-h stable baseline, we made a 6-h comparison after a 30-min intravenous endotoxin infusion (1 microg/kg) between six anesthetized control lambs and six anesthetized lambs in which the intravascular macrophages had been depleted 24 h previously. Three of the control lambs had been macrophage depleted and allowed to recover their intravascular macrophage population for >/=2 wk. After depletion, both the early and late pulmonary arterial pressure rises were dramatically attenuated. Our main interest, however, was in the acute lung microvascular injury response. The early and late rises in lung lymph flow and the increase in lung lymph protein clearance (lymph flow x lymph-to-plasma protein concentration ratio) were >90% attenuated. We conclude the pulmonary intravascular macrophages are responsible for most of the endotoxin-induced pulmonary hypertension and increased lung microvascular leakiness in sheep, although the unavoidable injury of other intravascular macrophages by the depletion regime may also contribute something.

Hepatic arterial infusion chemotherapy for liver metastases from breast cancer.

Between 1985 and 1992, 56 patients with unresectable liver metastases from breast cancer were treated by repeated hepatic arterial infusion chemotherapy employing an implantable port system. 5-Fluorouracil (5-FU) at 330 mg/m2 per week. Adriamycin (ADR) at 20 mg/m2 every 4 weeks, and mitomycin C (MMC) at 2.7 mg/m2 every 2 weeks were given to 42 patients. The remaining 14 patients received 5-FU at 330 mg/m2 per week and epirubicin (EPIR) at 20 mg/m2 every 2 weeks. As a rule, the treatment was performed on an outpatient basis. The side effects and complications observed included myelosuppression (41%), hepatic arterial occlusion (23%), and gastric mucositis (20%), but no major toxicity was encountered. The response rate (CR+PR) of the evaluated patients as determined from CT scans was 81%. The overall median survival period was 12.5 months. Only 14% of the patients died due to regrowth of liver metastases, and in 70% of the total cases, death due to liver metastases was avoided by this treatment. Thus, repeated hepatic arterial infusion chemotherapy for liver metastases from breast cancer might be capable of prolonging the survival of patients via avoidance of death due to the liver metastases.

Management of patients with unresectable liver metastases from colorectal and gastric cancer employing an implantable port system.

Between 1985 and 1990, 50 patients with unresectable liver metastases from colorectal cancer and 34 subjects with metastases from gastric cancer were treated by repeated hepatic arterial infusion chemotherapy employing an implantable prot system. A catheter was inserted into the hepatic artery via the left subclavian artery and was connected to the implantable injection port in each patient. 5-Fluorouracil (5-FU) at 330 mg/m2 per week (167 mg/m2 daily given continuously over the initial 3 months for colorectal cancer), Adriamycin (ADR) at 20 mg/m2 every 4 weeks and mitomycin C (MMC) at 2.7 mg/m2 every 2 weeks were given to all 34 patients with gastric cancer and to 31 of the colorectal cancer patients. The remaining 19 patients with colorectal cancer received 5-FU at 1,000 mg/m2 every week. As a rule the treatment was performed on an outpatient basis. The side effects and complications observed included myelosuppression (23%), hepatic arterial occlusion (21%), and gastroduodenal mucositis (12%), although no major toxicity was encountered. The response rate (CR+PR) among the evaluated patients as determined using CT scans was 67% for colorectal cancer and 73% for gastric cancer. The overall median survival was 12 months and 15 months, respectively. Good local control of liver metastases from the colorectal and gastric cancers was achieved by repeated hepatic arterial infusion chemotherapy employing an implantable port system without the need for hospitalization and without producing major toxicity. Thus, the implantable port system is very useful for the management of patients with unresectable liver metastases.

Significance of early measurement of serum hepatitis C virus RNA in predicting response to interferon therapy in patients with chronic hepatitis C.

BACKGROUND: Interferon can induce complete clearance of hepatitis C virus (HCV) in some patients with chronic hepatitis C. However, various side effects often require cessation of administration during the treatment period. Early prediction of response to interferon would be helpful. We evaluated measurement of serum HCV RNA 2 weeks after starting interferon as a predictor of response. METHODS: The presence of HCV RNA was measured in serum 2 weeks after starting therapy in 85 patients receiving natural interferon a (total 480 MU). RESULTS: Twenty-seven of 38 patients (71.1%) in whom serum HCV RNA had disappeared at 2 weeks achieved a sustained response. Only two out of 47 patients (4.3%) in whom serum HCV RNA had not disappeared at 2 weeks achieved a sustained response. Of 42 patients with pre-treatment HCV RNA concentrations less than 1 x 10(6) eq/ml, 26 of 30 patients (86.7%) whose HCV RNA had disappeared at 2 weeks achieved a sustained response, while only one of 12 patients (8.3%) whose HCV RNA was still detectable at 2 weeks had a sustained response. CONCLUSION: Clearance of serum HCV RNA after 2 weeks of treatment with interferon was more likely in patients with lower pre-treatment HCV RNA concentrations and they had a high likelihood of achieving a sustained response. In patients in whom serum HCV RNA was still detectable after 2 weeks of interferon therapy, a sustained response was most unlikely.

Purification of an antitumor-active, branched (1----3)-beta-D-glucan from Volvariella volvacea, and elucidation of its fine structure.

A (1--3)-beta-D-glucan branched by O-6 substitution (FCAP), obtained from the cold-alkali extract of the fruiting body of V. volvacea, exhibited potent growth-inhibitory activity against implanted tumors in mice. It contained protein and appeared to be heterogeneous. Fractionation by DEAE-Toyopearl column chromatography yielded an unbound, protein-free glucan fraction ([alpha]D - 30 degrees in M NaOH, mol. wt. 1.5-2 x 10(6)), which showed the highest antitumor activity. The polysaccharide had a moderately branched structure, consisting of a backbone chain of beta-(1--3)-linked-D-glucose residues, one out of five or six being substituted at O-6 with single glucosyl of beta-(1--6)-linked diglucosyl groups. Digestion of the glucan with exo-(1--3)-beta-D-glucanase yielded glucose and ++gentiobiose (molar ratio, 8:2:1.0), and a highly branched (d.b. 1/3), degraded glucan. Digestion with endo-(1--3)-beta-D-glucanase gave D-glucose, laminarabiose, a trisaccharide beta-D-Glcp-(1--6)-beta-D-Glcp-(1--3)-D-Glc, a tetrasaccharide beta-D-Glcp-(1--3)-beta-D-Glcp-(1--3)-[beta-D-Glcp-(1--6)]-D-Glc, and a highly branched (d.b. 1/2), enzyme-resistant glucan. The results suggest that the Volvariella glucan is structurally heterogeneous with regard to the distribution of branches, having less branched, moderately branched, and highly branched segments.

Preparation and characterization of antibodies against 6-O-alpha-D-xylopyranosyl-beta-D-glucopyranose (beta-isoprimeverose), the disaccharide unit of xyloglucan in plant cell-walls.

The p-aminophenyl beta-glycoside of 6-O-alpha-D-xylopyranosyl-D-glucopyranose (isoprimeverose), the disaccharide unit of plant xyloglucan, was coupled to bovine serum albumin, and the resulting glycoconjugate was used as an immunogen for the immunization of a rabbit. The immunochemical specificities of the rabbit antiserum raised against the glycoconjugate were characterized by immunodiffusion, quantitative precipitation, and hapten inhibition. After removal of anti-bovine serum albumin antibodies, the antiserum exhibited a specificity for the introduced disaccharide unit of the artificial antigen. The antibody-combining site was also shown to recognize the aglycon portion of the introduced hapten. The antiserum interacted with some xyloglucans, such as those from tamarind seed and the cell wall of pea stem. beta-Isoprimeverose and alpha-D-xylopyranosides were good inhibitors of the xyloglucan-antibody precipitation system, indicating that the antibodies recognize the beta-isoprimeverose unit of the xyloglucan.

Synthesis of three disaccharides for the preparation of immunogens bearing immunodeterminants known to occur on glycoproteins.

p-Nitrophenyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside was condensed with 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide, the product deprotected, and the disaccharide glycoside converted into p-trifluoroacetamidophenyl 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta- D-glucopyranoside. p-Nitrophenyl 3-O-benzoyl-4,6-di-O-benzylidene-alpha-D-mannopyranoside was condensed with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl bromide, and the product was deprotected, to yield p-nitrophenyl 2-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D-mannopyranoside. p-Nitrophenyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-beta-D-glucopyranoside was condensed with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide, and, after reduction, trifluoroacetylation, and deprotection, p-trifluoroacetamidophenyl 2-acetamido-2-deoxy-6-O-alpha-L-fucopyranosyl-beta-D-glucopyranoside was obtained.

Structure of an exocellular polysaccharide of Lactobacillus helveticus TN-4, a spontaneous mutant strain of Lactobacillus helveticus TY1-2.

Lactobacillus helveticus strain TN-4, a spontaneous mutant strain of Lactobacillus helveticus TY1-2, produced an exocellular polysaccharide from reconstituted skim milk. On the basis of the results of methylation analysis, enzymatic digestion, mild Smith degradation, mild acid hydrolysis, acetolysis, and 1D and 2D 1H-NMR spectroscopy, it was concluded that the polysaccharide has a D-galactofuranose containing hexasaccharide repeating unit with the following structure: [formula see text]

Structural study on an exocellular polysaccharide produced by Lactobacillus helveticus TY1-2.

Lactobacillus helveticus TY1-2 produced an exocellular polysaccharide when it was cultured in reconstituted skim milk. This polysaccharide is a high molecular weight heteropolymer of D-glucopyranosyl, D-galactopyranosyl, and 2-acetamido-2-deoxy-D-glucopyranosyl residues in the molar ratio 3.0:2.8:0.9. The primary structure of the polysaccharide was shown by glycose analysis, methylation analysis, Smith degradation, and NMR spectroscopy to be composed of branched heptasaccharide repeating units having the following structure: [formula: see text]

Synthesis of water-soluble, branched polysaccharides having D-mannopyranose, D-arabinofuranose, or oligo-D-arabinofuranose side-chains and their antitumor activity.

Branched polysaccharides having D-mannopyranose, D-arabinofuranose, or oligo-D-arabinofuranose side-chains were synthesized by the reaction of 3,4,6-tri-O-acetyl-(1,2-O-ethylorthoacetyl)-beta-D-mannopyranose, 3,5-di-O-benzoyl-(1,2-O-ethylorthobenzoyl)-beta-D-arabinofuranose, or 3-O-benzoyl-(1,2,5-O-orthobenzoyl)-beta-D-arabinofuranose with cellulose acetate or curdlan acetate, followed by desterification. The structure and antitumor activity of the water-soluble portion of the polysaccharides thus obtained were investigated. Polysaccharides synthesized from (1----3)-beta-D-glucan as the main chain with oligo-D-arabinofuranose side-chains exhibited high antitumor activity.

A case of intermittent bleeding Meckel's diverticulum.

The initial technetium-99m pertechnetate abdominal scintigraphy revealed equivocal or normal results. However, a second scintigraphy without pentagastrin demonstrated a focal area of persistently increasing radioactivity in the right lower quadrant of the abdomen. At surgery, Meckel's diverticulum was confirmed, and histological examination of the excised specimen revealed that it was lined with ectopic gastric mucosa. It has not been satisfactorily explained why the initial imaging failed to demonstrate the ectopic gastric mucosa. The necessity to perform repeated scintigraphy must be emphasized because 50 to 91 percent of bleeding Meckel's diverticula in the pediatric age group are said to contain gastric mucosa.

Gallium-67 citrate scintigraphy in the pre-operative evaluation of soft tissue tumors of the extremities.

We conducted a study of 90 patients with soft tissue tumor in their arms or legs, in order to determine the usefulness of scintigraphy with gallium-67 citrate as a diagnostic means. All patients had adequate scan images, and tumor tissues had been histologically confirmed by surgical resection. The subjects consisted of 19 patients with malignant tumors, 55 patients with benign tumors, and 16 patients with other disorders in which soft tissue tumor-like lesions occurred. When the activity of the tumor was more than the activity of the normal region of the contralateral extremity, it was estimated to be positive. The positive rate was found in 78% (15/19) of patients with malignant tumors, in 25% (14/55) of patients with benign tumors and in 31% (5/16) of patients with other disorders. Classified by diseases, high positive rates were observed in liposarcoma, leiomyosarcoma, malignant lymphoma, neurinoma, extra-abdominal desmoid and sarcoidosis. Out of 7 patients in which the activity of the tumor was equal to, or higher than that of the liver, 6 patients had malignant tumors and one patient was diagnosed as having an abscess. It seemed possible to distinguish between liposarcoma and lipoma by means of a gallium-67 scan. Furthermore, the gallium-67 scan was useful in detecting lesions of sarcoidosis as well as in evaluating the response to treatment.

Estimation of xyloglucan in vegetables by enzyme-linked immunosorbent assay.

We prepared the antibody specific against plant xyloglucan and applied it to enzyme-linked immunosorbent assay in order to estimate xyloglucan contents in several vegetables. In this experiment, xyloglucan was detected in the 24% aqueous potassium hydroxide-soluble fractions of tomato, cabbage, lettuce, and eggplant. The xyloglucan contents of these vegetables were estimated to be 1.4 mg, 54 micrograms, 14 micrograms, and 4 micrograms per one hundred grams of their edible portions, respectively, using tamarind xyloglucan as a standard xyloglucan.