Pharmacokinetics and enhanced PKR response in patients with chronic hepatitis C treated with pegylated interferon alpha-2b and ribavirin.

This study investigated the molecular and pharmacokinetic mechanisms of the enhanced antiviral efficacy associated with pegylated interferon (PEG-IFN) alpha-2b and ribavirin. The study involved comparing the expression of serial double-stranded RNA-activated protein kinase (PKR) before and during treatment in 26 PEG-IFN alpha-2b and 26 conventional IFN alpha-2b recipients matched for age, body weight and dose of ribavirin. The pharmacokinetics of PEG-IFN alpha-2b and ribavirin was analysed in 15 of the 26 PEG-IFN recipients. There was a rapid increase in PKR expression in both treatment groups, although expression from day 2 onwards was maintained at a significantly higher level in the PEG-IFN recipients (P < 0.05). C(max) of PEG-IFN occurred 12-48 h after the initial administration, with t(1/2) and C(min) being 49 h and 190 pg/mL, respectively. In contrast to ribavirin, accumulation of PEG-IFN was minimal. There was no association between serum PEG-IFN and ribavirin levels and virological response. Although baseline expression of PKR before treatment was marginally higher in nonresponders (NRs), from day 2 onwards, sequential PKR expression in response to PEG-IFN was higher in sustained viral responders compared with the NRs (P < 0.05). Significant correlations were found between kinetics of PKR expression and viral decline rates in each phase of hepatitis C virus dynamics (first phase, r = 0.67, P = 0.0006; second phase, r = 0.67, P = 0.001). In conclusion, improvement in pharmacokinetics following pegylation led to higher intracellular PKR expression, which was associated with enhanced virological efficacy of PEG-IFN-based combination therapy. The concentrations of both ribavirin and PEG-IFN alpha-2b were not associated with viral response and PKR expression.

Association of enhanced cyclooxygenase-2 expression with possible local immunosuppression in human colorectal carcinomas.

BACKGROUND: Prostaglandin (PG) E2 has an influence on antitumor lymphocyte reactions and causes local immunosuppression at tumor sites. The contribution of cyclooxygenase (COX), a key enzyme in PGE2 synthesis, to this effect is still unclear. We examined if cyclooxygenase (COX)-2 is involved in local immunosuppression in human colon carcinoma cell lines and in clinical tumor specimens. METHODS: PGE2 concentrations were measured in culture media from a highly COX-2-expressing human colon carcinoma cell line (CE-1) and other cell lines. Lymphocyte proliferation in response to a mitogen was used to evaluate immunosuppression in tumor cell-lymphocyte cocultures with and without selective COX-2 inhibitor NS-398. We also evaluated expression of COX-2 mRNA in surgical specimens of colorectal carcinoma by reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein by immunohistochemistry, correlating COX-2 expression with clinicopathologic features. RESULTS: CE-1 cells produced large amounts of PGE2, which was significantly inhibited by NS-398. The proliferation index of lymphocytes cocultured with CE-1 cells was significantly less than that of control lymphocytes; again, this effect was inhibited by NS-398. While human colorectal carcinoma tissue expressed more COX-2 mRNA and protein than nonneoplastic tissue, no significant correlation was found between COX-2 levels and clinicopathologic features. CONCLUSIONS: Overexpression of COX-2 in colon cancer may cause local immunosuppression, and COX-2 inhibitors might be therapeutically useful against these tumors.

Detection of metastatic breast cancer by beta-hCG polymerase chain reaction.

Reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of occult malignancies in breast cancer patients is evolving as a useful diagnostic tool. However, no reliable molecular mRNA markers are available. We developed an RT-PCR plus Southern blot assay using beta-hCG (beta-subunit of human chorionic gonadotropin) gene expression as a tumor marker for detection of breast malignancies metastatic to tumor-draining lymph nodes and blood. Breast carcinoma cell lines, primary breast malignancies and human placenta were used as positive controls for establishing the beta-hCG RT-PCR assay. Peripheral blood leukocytes (PBL) from normal volunteer donors, normal breast tissue and lymph nodes from cancer-free patients were used as negative controls. beta-hCG RT-PCR was used to assess tumor cell presence in PBL and tumor-draining axillary nodes from patients with AJCC stage I-IV breast cancer. The assay sensitivity and specificity were enhanced by restriction endonuclease digestion of an Sty I site of the RT-PCR cDNA product followed by Southern blot analysis. beta-hCG mRNA was expressed in all breast cancer cell lines and 80% of primary breast cancers; it was not expressed in negative controls. The assay reliably detected one cancer cell in > 10(7) PBL, with a sensitivity of 10(-5) microgram RNA. Eighty percent of PBL and 61% of tumor-draining axillary nodes from breast cancer patients expressed beta-hCG mRNA. The assay is a sensitive and specific method of identifying breast cancer cells in breast tissues, lymph nodes and blood.

Interleukin 4 inhibits hepatocyte growth factor-induced invasion and migration of colon carcinomas.

Hepatocyte growth factor (HGF) is known to have a number of biological properties including promoting tumor progression of human carcinomas. Metastasis involves a number of events that are attributed to induction by paracrine factors such as HGF. Identification of natural inhibitors of these events would allow better control of tumor progression. Recently we demonstrated that interleukin 4 (IL-4) can regulate proliferation of various human carcinoma cell lines. In the present study, we used established human colon carcinoma cell lines and primary colon carcinoma cell cultures to determine if IL-4 could regulate HGF-induced cell proliferation and other events of tumor progression such as MMP (matrix metalloproteinases)-1, -2, and -9 production, cell migration and cell-matrix invasive activity. All colon carcinoma cell lines expressed HGF and IL-4 receptors. IL-4 significantly inhibited HGF-induced proliferation of one cell line. Cell-matrix invasion was significantly enhanced by HGF (0.1-10 ng/ml); IL-4 (1-10 U/ml) significantly inhibited HGF-induced invasion in a dose-dependent manner. IL-4 also inhibited HGF-induced cell-matrix invasion of metastatic colon carcinoma cells and HGF-induced cell migration. HGF enhanced MMP-1, -2, and -9 production by cell lines. This effect could be inhibited by IL-4. These findings indicate that IL-4 is a potent inhibitor of HGF-induced invasion and metastasis-related functions of human colon carcinoma cells.

Detection of beta-human chorionic gonadotropin mRNA as a marker for cutaneous malignant melanoma.

The beta chain of human chorionic gonadotropin (hCG) hormone is produced by fetal cells, gonadal cell tumors and several types of non-gonadal carcinoma. hCG is composed of an alpha and a beta chain, the latter of which can be used to distinguish the molecule from other related gonadotropin hormones. Detection of beta-hCG mRNA transcripts can be potentially useful as a marker to identify tumor cells. We devised a highly specific and sensitive assay to detect the atavistic expression of beta-hCG in cutaneous melanoma by RT-PCR. Twenty-four melanoma cell lines and 43 melanoma biopsies were evaluated for beta-hCG mRNA expression. An RT-PCR assay was developed to specifically distinguish beta-hCG poly-A mRNA from other related gonadotropin beta chains. This was performed by endonuclease digestion of a unique Sty 1 site in the beta chain, followed by Southern blot analysis with a beta-hCG cDNA probe. Of the 24 melanoma cell lines analyzed, 18 expressed beta-hCG mRNA. Analysis of melanoma biopsy specimens revealed beta-hCG mRNA expression in 17/25 melanoma-positive TDLN, and in only 5/15 non-lymphoid melanoma metastases. Beta-hCG mRNA expression had a 53% correlation to tyrosinase mRNA, a predominant melanoma marker. Beta-hCG mRNA was not detected in normal donor PBL and normal lymph nodes. Detection of beta-hCG mRNA expression may be a useful molecular marker to define a subset of malignant melanoma.

Effects of granulocyte colony stimulating factor and monobactam antibiotics (Aztreonam) on neutrophil functions in sepsis.

Possible clinical use of a recombinant human granulocyte colony stimulating factor (rG-CSF) and a newly developed monobactam antibiotics (Aztreonam) for the treatment of gram-negative sepsis was investigated. Gram-negative sepsis was induced in male WKA rats by cecal ligation and puncture (CLP). Untreated CLP rats all died by septicemia with severe peripheral blood leukocytopenia within 5 days after the operation. When we administered 2.0 micrograms/kg of rG-CSF and/or 20 mg/kg of Aztreonam intravenously just after the operation, the rats survived longer than the untreated CLP rats. These drugs were found to be more effective when used in combination. Since these rats showed an increase in leukocyte counts, we next examined the changes in the functions of polymorphonuclear leukocytes (PMNs, mainly neutrophils) after the treatment. PMNs from untreated CLP rats at 24 hr after the operation exhibited enhanced plastic-dish adherence, suppressed chemotaxis, and depressed O2 production when compared with PMNs from control animals. A single injection of rG-CSF restored both the depressed chemotaxis and the O2 production to levels greater than those of controls. Although a single injection of Aztreonam could not improve the suppressed O2 production, it could restore the depressed chemotaxis. Interestingly, simultaneous injection of Aztreonam with rG-CSF significantly enhanced the effect of rG-CSF on the PMN functions. These data suggest that the Aztreonam and rG-CSF may be useful for the treatment of gram-negative sepsis, especially when used in combination.

Potential role of hepatic macrophages in neutrophil-mediated liver injury in rats with sepsis.

We investigated the pathogenesis of septic liver injury in rats caused by cecal ligation and puncture. In this model, numerous neutrophils accumulated in the liver in parallel with the development of liver dysfunction. The supernatants of hepatic macrophages isolated from these septic rats 24 hr after cecal ligation and puncture had enhanced chemotactic activities for human neutrophils. These results suggest that in sepsis, hepatic macrophages attract neutrophils to the liver. Human neutrophils preincubated in this macrophage supernatant had the following biological activities not seen in the sham-operated controls. (a) They became more adherent to cultured endothelial cells through up-regulation of adhesion molecules such as CD11b/CD18, (b) their chemiluminescence was markedly elevated. These functional changes of cecal ligation and puncture hepatic macrophages were the same as those in endotoxin-pretreated hepatic macrophages after isolation from normal rats. Therefore we suspect that hepatic macrophages are activated by portal vein endotoxin in sepsis. These activated hepatic macrophages secreted chemical mediators of inflammation, including leukotriene B4 and tumor necrosis factor. In conclusion, hepatic macrophages seem to interact closely with neutrophils and play an important role in the pathogenesis of septic liver injury.

Effect of dietary fat and cholesterol on dimethylbenz[a]-anthracene-induced mammary tumorigenesis in Sprague-Dawley rats.

The effect of dietary fats and cholesterol on dimethylbenz[a]-anthracene-induced mammary tumorigenesis was studied in female Sprague-Dawley rats. When the dietary fat source (at the 5% level) was palm oil (saturated fat) or corn oil (unsaturated fat), dietary cholesterol at the 0.2% level increased the tumor number of rats fed corn oil, but not those fed palm oil. Perilla oil (rich in alpha-linolenic acid) reduced tumor development as compared with safflower oil (rich in linoleic acid), but again dietary cholesterol at the 0.5% level diminished the favorable effect of n-3 polyunsaturated fatty acid (PUFA). The adverse effect of cholesterol was also observed in the n-6 PUFA fat. The promotive effect of dietary cholesterol was not necessarily associated with the change in the production of prostaglandin E2 by the tumor tissue or in the immunopotentiation. These results at least stress that the contrasting effects of dietary fats should be carefully evaluated whether cholesterol is present simultaneously or not.

Suppressive effect of sesamin against 7,12-dimethylbenz[a]-anthracene induced rat mammary carcinogenesis.

The effects of dietary supplementation of sesamin on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in female Sprague-Dawley rats were studied. Experimental diets containing 0.2% sesamin (an equiweight mixture of sesamin and episesamin) or 0.2% alpha-tocopheryl acetate were given to rats starting 1 week before intragastric administration of DMBA (10 mg/rat). Sesamin significantly (p less than 0.05) reduced the cumulative number of palpable mammary cancers by 36% at 12 weeks post-DMBA administration compared with animals on a control diet. Alpha-tocopheryl acetate inhibited both the incidence and the cumulative number of mammary tumors by 20% and 45%, respectively. Concentrations of lipid peroxides in plasma, liver and tumors were all decreased in both sesamin and alpha-tocopheryl acetate groups. The activity of peripheral blood mononuclear cells (PBMC) increased in rats fed sesamin (140 to 150% of the control and alpha-tocopheryl acetate groups). Fatty acid compositions of plasma, liver and tumor phosphatidylcholine showed a decreased tendency of the metabolism of linoleic acid to arachidonic acid and hence of the plasma concentration of prostaglandin E2 in the sesamin group. The inhibitory effect of sesamin on DMBA-induced mammary carcinogenesis may be ascribed, at least in part, to immunopotentiation and increased antioxidative activity.

Pilonidal sinus on the neck.

A recurrent nuchal abscess was treated in a 21-year-old obese young man with a total excision of the lesion. In the histopathological findings, many similarities were found between this lesion and pilonidal sinus. We discuss the pathogenesis of this lesion, as well as our belief that this case was a rare example of pilonidal sinus on the neck.

[Immunological assessment of the pathogenesis of septic-MOF: relationship between complement activation and changes in neutrophil functions].

We investigated the pathogenesis of septic-MOF through the relationship between changes in neutrophil functions and degree of complement activation. The patients' neutrophils exhibited enhanced adherence to HUVEC, suppressed chemotaxis toward C5a, enhanced production of oxygen radicals and lysosomal enzymes. These changes in neutrophil functions related to complement activation elicited via classical pathway. Moreover, the activated complement participated in tissue injuries due to the cytolytic action of the terminal complement complexes such as membrane attack complex (MAC). In conclusion, the combination of neutrophil and complement was strongly associated with the pathogenesis of the septic-MOF.

Multiple forms and glycoprotein nature of acid phosphatase, alpha-fucosidase and alpha-mannosidase of psoriatic scales.

Isoelectric focusing and concanavalin A-Sepharose chromatography were used to study the multiple forms and glycoprotein natures of so-called lysosomal hydrolases from psoriatic scales. Acid phosphatase appeared as 5 different forms with pI values of 6.5, 6.1, 5.8, 5.6 and 5.45. Seven isoenzymes of alpha-fucosidase were identified with pI values of 6.4, 6.2, 5.9, 5.75, 5.65, 5.4 and 5.2. Acid alpha-mannosidase activity appeared as one peak with pI value of 6.75 and a weak activity of neutral alpha-mannosidase was present with pI value of 6.7. Incubation of the extract with neuraminidase increased their pI values of acid phosphatase, alpha-fucosidase and alpha-mannosidase to the more basic forms. This finding suggests that epidermal acid phosphatase, alpha-fucosidase and alpha-mannosidase have some N-acetylneuraminic acid residues. In addition concanavalin A-Sepharose column chromatography was also performed to confirm the glycoprotein nature of acid phosphatase. This enzyme was bound to the column and not released from the column even with the treatment of 0.5 M NaCl, but the enzyme was eluted from the column with the treatment of alpha-methyl-D-glucoside.

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells.

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin. Following supplementation with saturated fatty acids of longer than 15 carbons (100 micron) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth. Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35--41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72--82% after addition of unsaturated fatty acids. A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids. The results suggest that maintenance of membrane fluidity by unsaturated fatty acids in membrane phospholipids is critical to membrane integrity and cell growth.