Augmented anti-tumour effect of lipopolysaccharide with G-CSF without enhancing body weight loss in mice bearing MH134 hepatoma.

BACKGROUND: Although lipopolysaccharide (LPS) is reported effective for cancer, developmental application as anti-tumour therapeutic agents have been prevented due to its severe toxicity. Since antitumour action of LPS was suggested to be partly related to the function of neutrophil, we investigated the effects of granulocyte colony stimulating factor (G-CSF), a cytokine enhancing neutrophil function, on antitumour activity of bacterial LPS against a murine syngeneic hepatoma MH134. METHODS: Effect of G-CSF (30 µg/kg i.v. pretreatment, 4 days) and LPS (20µg/mouse) on tumor growth and survival days of mice bearing MH134 hepatoma were monitored. RESULTS: 4 day treatment of G-CSF 30 µg/kg increased the neutrophil level with statistical significance. On the MH134 hepatoma bearing mice, LPS significantly inhibited the tumour growth. Although G-CSF pretreatment alone did not inhibit the growth, once combined with LPS, significant inhibition was observed compared with LPS group. Tumour regression was demonstrated in combination group (6/12 mice), and survival of the mice in the combination group exceeded those of the LPS monotherapy group without enhancing the body weight loss. CONCLUSIONS: Combination of G-CSF and LPS prominently inhibit the tumour growth and elongated the survival days without enhancing a toxic response to LPS treatment.

Effects of Liposome-Entrapped Muramyl Tripeptide Phosphatidylethanolamine (L-MTP-PE) on the Tumor Growth and Survival of Mice Bearing Syngeneic Tumor in Combination with a Chemotherapeutic or Immunomodulatory Agent.

Antitumor activities of L-MTP-PE (Liposome entrapped myuramyl tripeptide phosphatidylethanolamine) in the combination treatment with chemo- or immune-therapeutic antitumor agents against various syngeneic tumors were tested.Against Meth A fibrosarcoma solid tumor system, L-MTP-PE showed slight but statistically significant elongation of survival days against 5-FU monotherapy in spite of its non-effect on tumor growth, when combined with 5-FU. Against liver metastasis model of M5076 carcinoma, L-MTP-PE showed a tendency of elongation of survival days by its single drug treatment, however, elongation with statistical significance was observed in the combination treatment with 5-FU in comparison with control group.These data suggest that L-MTP-PE seems to elongate the survival days of the solid tumor bearing mice and the liver metastasis model basically due to its saving effect on chemotherapeutic drug-induced immunosuppression. In the combination with an immunotherapeutic agent in mice, TNF production induced by another biological response modifier OK-432 was potentiated when primed with L-MTP-PE. L-MTP-PE also potentiate the antitumor effect of OK-432 possibly through the enhanced production of TNF-α. Combination of L-MTP-PE and OK-432 is considered to be a candidate for a new treatment model for cancer.

Different activities of antitumor immunomodulators to induce neutrophil adherence response.

Functions of neutrophils, major participant in host defense mechanisms, are known to be regulated by various types of immunomodulators. Capacity of immunomodulators which are reported to show antitumor effect in vivo to induce neutorophil adherence response in vitro was investigated. Several bacterial immunomodulators (OK-432, Corynebacterium parvum, B.C.G.) and components of bacteria cell walls (lipopolysaccharide (LPS), lipid A, lipoteicoic acid, N-cell wall skelton (N-CWS), muramyl dipeptide (MDP)) and fungal polysaccharides (lentinan, zymosan A, etc.) were tested. Neutrophils prepared from peripheral blood of healthy men were incubated with each immunomodulator at 37°C for 60 min in 96 well plastic plates, then neutrophils adherent to substratum were stained by crystal violet and their optical density at 570 nm was measured as a parameter of neutrophil adherence. Although purified polysaccharides mainly prepared from fungi did not induce the adherent response, not only bacterial bodies and their components but also tumor necrosis factor-α (TNF-α) clearly induced it. On the base of these results, functional classification and typing of immunomodulators by different activities in neutrophil adherence was discussed.

Increase in hydrophobicity of Bacillus subtilis spores by heat, hydrostatic pressure, and pressurized carbon dioxide treatments.

The effects of heat treatment (HT), hydrostatic pressure treatment (HPT), and pressurized carbon dioxide treatment (CT) on surface hydrophobicity of B. subtilis 168 spores were investigated. The spore surface hydrophobicity was measured by determining the ratio of hydrophobic spores (RHS) that were partitioned into the n-hexadecane phase from the aqueous spore suspension. The RHS after HT generally increased in a temperature-dependent manner and reached approximately 10% at temperatures above 60°C. The effects of pressurization by HPT and accompanying temperature on increased RHS were complex. The highest RHS after HPT was approximately 17%. Following CT, RHS reached approximately 80% at 5 MPa at 80°C for 30 min. An increased treatment temperature enhanced RHS by CT. The increase in RHS by CT led to the formation of spore clumps and adhesion of spores to hydrophobic surfaces. Acidification of spore suspension to pH 3.2, expected pH during CT, by HCl also increased the adhesion of spores at the similar degree with CT. The spore surface zeta potential distribution was not changed by CT. Furthermore, spores with increased RHS after CT had germination-like phenomena including loss of their refractility and enhanced staining by 4',6-diamidino-2-phenylindole. Physiological germination that was induced by the addition of l-alanine also increased the RHS. From these results, it is clear that CT under heating considerably increases RHS. CT under heating considerably increases RHS. This increase in RHS may be due to acidification or germination-like phenomena during CT.

Sublethal high hydrostatic pressure treatment reveals the importance of genes coding cytoskeletal protein in Escherichia coli morphogenesis.

We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, ΔzapA and ΔrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.

An attempt to discriminate between the hydrophobic and aromatic pi-pi interactions in the copper(II) ternary complexes CuLA with L=1,10-phenanthroline or 2,2'-bipyridyl and A=para-X-substituted phenylalaninates.

For the title complexes, the value of formation constant K(CuL+A) is higher than that of K1(CuA2). According to the mechanistic consideration, log K(CuL+A) is calculated for regular Cu(II) complexes with neither special enhancement nor diminution of the stability constant. Then, the difference of log K(CuL+A)(obs)-log K(CuL+)(calc) represents extrastabilization due to the hydrophobic interactions and the aromatic pi-pi interactions. The former has been found to be proportional to the free energy of the transfer of side chains of aminocarboxylates A. The discrimination between the hydrophobic and the aromatic pi-pi interactions has been attempted.

Lamivudine therapy for hepatitis B virus reactivation in a patient receiving intra-arterial chemotherapy for advanced hepatocellular carcinoma.

We describe a case of hepatitis B virus (HBV) reactivation that responded to lamivudine therapy in a 58-year-old man with advanced hepatocellular carcinoma (HCC). After seroconversion of hepatitis B e antigen to e antibodies by interferon therapy, the patient was found to have HCC with a portal tumor thrombus. A transarterial port was placed in the right femoral artery to permit infusion of epirubicin and cisplatin. After 3 months of arterial chemotherapy, the serum alpha-fetoprotein level had decreased and tumor staining diminished. Laboratory examinations suggested a flare-up of hepatitis B. Lamivudine was given to manage HBV reactivation. After 1 month, the serum HBV DNA level fell below the detection limit, and the alanine aminotransferase activity decreased to the normal range. With further arterial chemotherapy for HCC, no tumor staining was detected on computed tomography. Administration of lamivudine decreased serum HBV DNA levels for 7 months. Our findings suggest that HBV may be reactivated during chemotherapy for HCC, similar to other types of malignancies, and that lamivudine is effective for the management of HBV reactivation.

Association of HLA alleles with response (especially biochemical response) to interferon therapy in Japanese patients with chronic hepatitis C.

The response of chronic hepatitis C to interferon (IFN) treatment is classified as complete response (CR), biochemical response (BR), or no response (NR). Several studies have found no difference in prevention of hepatocellular carcinoma by IFN therapy between patients with CR and those with BR. We investigated whether specific human leukocyte antigen (HLA) alleles were associated with response to IFN, especially BR, in 138 patients with chronic hepatitis C. Comparing patients with and without CR, male, a low viral titer, genotype 2a or 2b, HLA-B55, and HLA-DRB1-0803 were more common in the group with CR. Multivariate analysis showed that age (adjusted odds ratio [OR], 0.95 by every year [95% confidence interval [CI] 0.90 - 0.99], p = 0.028), genotype 2a or 2b (5.21 [95% CI 1.63 - 16.6], p = 0.005), and low viral titer (8.58 (2.66 - 27.7), p < 0.001) were associated with CR. Comparing patients with BR and NR, the pretreatment alanine aminotransferase (ALT) level was lower in the BR group (p < 0.001). Both HLA-B7 and HLA-DRB1-0101 were more common in this group (p = 0.002). As the alleles HLA-B7 and HLA-DRB1-0101 were in linkage disequilibrium, the HLA-B7-DRB1-0101 haplotype may be associated with BR. Multivariate analysis indicated that a low ALT level (0.98 by every 1 IU/L [95% CI 0.98 - 0.99], p = 0.001) and HLA-B7-DRB1-0101 haplotype (32.3 [95% CI 1.50 - 693.1], p = 0.026) contributed significantly to BR. This study suggested that host HLA expression, but not viral factors, can influence BR.

Changes in hypervariable region 1 in patients with chronic hepatitis C of genotype 1b with biochemical response to interferon.

BACKGROUND/AIMS: The outcome of interferon (IFN) therapy of hepatitis C virus (HCV) infection can be classified as a complete viral response (CR), biochemical response (BR), or no response (NR). Why alanine aminotransferase (ALT) activity decreases in patients with BR despite viral persistence is unknown. METHODS: Of 158 patients infected with HCV genotype 1b, all 20 patients with BR and 20 of the 114 patients with NR, matched for viral load to the BR group, were studied. We sequenced nucleotides in the hypervariable region (HVR) of serum HCV RNA, and analyzed quasispecies of this region by the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP). RESULTS: In HVR 1, SSCP patterns differed after therapy; the major clone before therapy disappeared with therapy in eight of the 20 BR patients, but in none of the 20 NR patients (P=0.0033; Fisher's exact test). PCR products of HVR 1 from six patients were cloned before and after therapy, and 40 clones from each patient were sequenced each time. Results of cloning and sequencing were generally consistent with those of SSCP. For the six patients, a major clone could be identified both before and after therapy. In two patients with BR, there were many changes in the amino acid sequence of the major clone after IFN; in one patient with NR, mutations were not found. CONCLUSION: Changes in the major viral clone with IFN treatment may be related to the decrease in ALT activity in some patients, in spite of the continued presence of HCV RNA.

Accurate prediction of response to interferon therapy by repeated measurement of hepatitis C virus core protein in patients with chronic hepatitis C.

OBJECTIVE AND METHODS: Serum levels of hepatitis C virus (HCV), a predictor of the response to interferon (IFN) therapy, can fluctuate widely in patients with chronic hepatitis C, even without antiviral therapy. In order to increase the accuracy of predicting the response to therapy, serum samples from 134 patients with chronic hepatitis C were collected twice: 1.0-4.5 months before and just before the start of IFN therapy, and were tested for HCV core protein by a fluorescent enzyme immunoassay. RESULTS: Forty-one (31%) patients had a complete response to IFN and 93 (69%) had no response. The most useful cutoff value between high and low viral loads for predicting the response to therapy was 40 pg/ml of HCV core protein. A complete response was obtained more frequently in 32 of 45 patients with persistently low viral loads than in those with persistently high viral loads (7 of 71) (p < 0.0001). In 2 of 18 patients with a low viral load at one time point but a high viral load at another, the rate of complete response was similar to that in patients with persistently high viral loads. CONCLUSION: Prediction of the response to IFN therapy based on HCV core protein measurement at two time points before therapy is more reliable than that based on HCV core protein measurement at only one time point.

Dynamics of hepatitis C virus monitored by real-time quantitative polymerase chain reaction during first 2 weeks of IFN-beta treatment are predictive of long-term therapeutic response.

The relation between the change in hepatitis C virus (HCV) RNA levels at the start of interferon-beta (IFN-beta) treatment and the long-term therapeutic response remains poorly defined. In 20 patients with chronic hepatitis C who received IFN-beta (total dose 126-756 MU), the changes in serum HCV RNA during the first 2 weeks of therapy were monitored by real-time quantitative polymerase chain reaction (PCR). The serum HCV RNA level decreased rapidly during the first 24 h of therapy (first phase) and more slowly thereafter (second phase), with a mean exponential decay rate of 1.17 log10/day and 0.37 log10/day, respectively. Three patients had a sustained virologic response, 10 patients had a transient response, and 7 patients had no response. The differences in the rate of first-phase viral decline among the three groups were not significant (p = 0.21), but the differences in the rate of second-phase viral decline were significant (p = 0.0021). The mean decay rate between the end of the first 24 h and day 14 was 0.96 +/- 0.43 log10/day in sustained responders, 0.39 +/- 0.30 log10/day in transient responders, and 0.13 +/- 0.09 log10/day in nonresponders. We conclude that during the first 2 weeks of therapy, changes in serum HCV RNA levels as monitored by real-time quantitative PCR can be used to predict the long-term response to treatment with IFN-beta.

Changes in serum levels of hepatitis C virus genotype 1b monitored by real-time quantitative polymerase chain reaction as a predictor of long term response to interferon-alpha treatment.

OBJECTIVE: The aim of this study was to find whether there is a relationship between the changes in the amounts of hepatitis C virus (HCV) at the start of interferon treatment and the long term response to therapy. METHODS: In 20 patients with HCV genotype 1b each given 880 MU of interferon-alpha, the changes in serum HCV RNA during the first 2 wk of therapy were monitored by real-time quantitative polymerase chain reaction (PCR). RESULTS: Real-time quantitative PCR detected HCV RNA at 10(1)-10(8) copies/ml. Serum HCV RNA decreased rapidly between 8 and 24 h after the first administration (first phase) and more slowly thereafter (second phase), with median exponential decays of 2.14 and 0.11 log10/day, respectively. Four patients had sustained virological responses, nine patients had transient responses, and seven patients had no responses. The differences in the rate of first-phase viral decline among the three groups were not significant (p = 0.34), but the differences in the rate of second-phase viral decline were significant (p = 0.0004); the median viral decline (interquartile range) in the second phase was 0.48 (0.42-0.50) log10/day in patients with sustained responses, 0.16 (0.10-0.19) log10/day in patients with transient responses, and 0.026 (0.017-0.040) log10/day in patients with no responses. CONCLUSIONS: Changes in serum levels of HCV genotype 1b in the first 2 wk of interferon-alpha treatment, monitored by real-time quantitative PCR, can be used for prediction of the long term therapeutic response.

GB virus C and TT virus infections in Japanese patients with autoimmune hepatitis.

The association of the newly identified viruses, GB virus C (GBV-C) and TT virus (TTV), with autoimmune hepatitis remains to be elucidated. Sera from 20 Japanese patients with autoimmune hepatitis and 50 volunteer blood donors were assayed for GBV-C RNA, antibodies to the GBV-C second envelope protein (E2), and TTV DNA. GBV-C RNA was examined by reverse-transcription polymerase chain reaction (PCR). Anti-GBV-C E2 (a marker of past infection) was tested by an enzyme-linked immunosorbent assay. TTV DNA was amplified by PCR using two different sets of primers: one derived from the original N22 sequence (Set A) and the other from the untranslated region (Set B). None of the patients or controls had GBV-C RNA. Anti-GBV-C E2 was found significantly more often in patients with autoimmune hepatitis (3/20) than in controls (1/50; P = 0.034). The prevalence of TTV DNA detected by primers Set A and that detected with either Set A or B were similar among patients with autoimmune hepatitis (4/20 and 16/20, respectively) and controls (9/50 and 40/50, respectively). Clinical characteristics did not differ in association with any of these viral markers. Of the 13 TTV isolates amplified with Set A, seven were classified as genotype 1a, four as genotype 1b, and 2 as genotype 3; no particular strain was associated with autoimmune hepatitis. These findings provide no compelling evidence that GBV-C or TTV has a pathogenic role in autoimmune hepatitis.