Oral-mucosal administration of IFN-alpha potentiates immune response in mice.

We studied the effects of oral-mucosal administration of murine interferon-alpha (Mu-IFN-alpha) on immune responses and infection with vaccinia virus (VV) in mice. When Mu-IFN-alpha was administered to sheep red blood cell (SRBC)-sensitized mice for 4 or 5 days, Mu-IFN-alpha significantly enhanced delayed-type hypersensitivity (DTH) and antibody production, with maximum enhancement of each at 1 IU/body. To investigate the antiviral effect of oral-mucosal Mu-IFN-alpha, mice were infected with VV, and Mu-IFN-alpha was administered for 15 days. Pocks were observed in the tail skin of infected mice, and Mu-IFN-alpha at doses of 1, 10, and 100 IU/body significantly suppressed pock formation. Also, VV-specific cytotoxic T cells (CTL) were observed in the spleen from the same mice at 7 days after infection, and Mu-IFN-alpha enhanced CTL activity at doses above 1 IU/body. These results suggest that the oral-mucosal Mu-IFN-alpha may have potentiating effects on cellular and humoral immune responses, which may contribute to its effects against VV.

Design of variants of the second domain of urinary trypsin inhibitor (R-020) with increased factor Xa inhibitory activity.

The second domain (R-020) of human urinary trypsin inhibitor (UTI) exerts similar inhibitory activities on trypsin, alpha-chymotrypsin, leukocyte elastase, and plasmin to those of UTI itself, and additionally inhibits coagulation factor Xa (FXa) and plasma kallikrein, on both of which UTI has no inhibitory effect. In the present study, we attempted to increase this FXa-inhibitory activity by modeling the structure of R-020-FXa complex and substituting one or two amino acids in R-020 using recombinant DNA technology. Molecular modeling of R-020 and FXa was performed with reference to X-ray analysis of the complex of bovine pancreatic trypsin inhibitor (BPTI) and bovine trypsin to determine the site of amino acid modification. The expression plasmids into which R-020 genes with base substitution were inserted were prepared and introduced into Escherichia coli to express R-020 variants. The resulting variants were purified and their enzyme inhibitory activities were measured. The FXa-inhibitory activity was increased in four variants with single amino acid substitution. With another four variants having two amino acid substitutions involving combinations of the above single amino acid substitutions, the FXa-inhibitory activity was further increased. Because the electrostatic interaction within R-020-FXa complex seemed stronger in these R-020 variants, this increase in FXa-inhibitory effect was speculated to be a consequence of more potent binding between the enzyme and the inhibitor.

[Concentrations of cefmenoxime in human bone marrow blood and osseous tissue].

One gram of cefmenoxime (CMX) was administered to each of 10 patients by a one shot intravenous injection at the beginning of surgery. At 30, 60, 90, 120 minutes after the administration, concentrations of CMX in venous blood, bone marrow blood and bone tissue were assayed by the agar-well method. CMX was smoothly transmigrated after the injection from venous blood to bone marrow blood and bone tissue, and effective concentrations higher than MIC80 values were maintained for several hours. CMX, therefore, appears to be a useful drug for the prophylaxis and the treatment of postoperative infections.

[Basic study on human interferon-beta: Part I. Antiviral effect].

A study was performed on the antiviral effects of human interferon-beta (IFN-beta, MR-21) produced by a normal human diploid fibroblast cell strain. Of 4 DNA and 9 RNA viruses used in this study, all were inhibited by the IFN-beta and the IC90S obtained were approximately 10(1)-10(3) IU/ml. Results showed that IFN-beta had a very wide antiviral spectrum. The IFN-beta was able to induce 2',5'-oligo A synthetase activity in RSa, HeLa S3 and M08 cell lines but not in HEC-1C cell line lacking receptors for IFN-beta. This strongly suggests that one of the antiviral mechanisms might function through the induction of 2',5'-oligo A synthetase activity through the IFN-beta receptor.

[Basic study on human interferon-beta: Part II. Anticellular effect].

The anticellular effect of human interferon-beta (IFN-beta, MR-21) was examined in vitro. IFN-beta had a wide anticellular spectrum in proliferation inhibition assay. Of 67 human cell lines including 62 tumor-originated cell lines, 51 were sensitive to IFN-beta (IC50 less than 1,000 IU/ml) while 16 including normal diploid cells were resistant (IC50 greater than or equal to 1,000 IU/ml). These anticellular effects were reversible and there was no relationship between the origins of tumor cell lines and their sensitivity to IFN-beta. IFN-beta induced not only proliferation inhibition but also cytotoxicity in several cell lines. It was suggested that the mode of anticellular effects of IFN-beta was mainly time-dependent.

[Basic study on human lymphoblastoid interferon-alpha (MOR-22): Part I. Antiviral effect].

A study was performed on the antiviral effects of human interferon-alpha (MOR-22) produced by a human lymphoblastoid cell line (BALL-1). Of 4 DNA and 9 RNA viruses used in this study, all were inhibited by the IFN-alpha and the IC90s obtained were 10(1)-10(4) IU/ml. Results showed that IFN-alpha had a very wide antiviral spectrum. The IFN-alpha was able to induce 2',5'-oligo A synthetase activity in RSa, HeLa S3 and M08 cell lines but not in a HEC-IC cell line lacking receptors for IFN-alpha. This strongly suggests that one of the antiviral mechanisms might function through the induction of 2',5'-oligo A synthetase activity through the IFN-alpha receptor.

[Basic study on interferon-beta: Part IV. Antitumor effect on nude mouse-transplanted human tumors].

The effects of human interferon-beta (IFN-beta, MR-21) on the growth of xenografted human tumors in nude mice were examined. IFN-beta was administered to mice with malignant melanoma (SK-MEL-28 and Sk-14) intratumorally at a dose of 1 X 10(5)-3 X 10(5) IU/mouse, with acute leukemia (CCRF-HSB-2) intratumorally at a dose of 3 X 10(5) IU/mouse, with glioblastoma (U-373 MG) intravenously or intratumorally at a dose of 1 X 10(5)-6 X 10(5) IU/mouse, or with uterine cervical tumor (HeLa S3) intravenously at a dose of 0.3 X 10(5)-1 X 10(5) IU/mouse. IFN-beta inhibited the growth of all of these tumors in a dose-dependent manner.

[Basic study on human interferon-beta: Part III. The mechanisms of its antitumor effect].

The mechanisms of the direct and indirect antitumor effects of human interferon-beta (IFN-beta, MR-21) were examined. IFN-beta suppressed DNA, RNA and protein synthesis in cells derived from human tumor. The expression of cellular oncogenes (c-Ha-ras and c-myc) in tumor-originated cells was also suppressed by IFN-beta. These results suggest that such suppression is one possible mechanism of the direct anticellular effect induced by IFN-beta. IFN-beta augmented NK cell activity and the ADCC activity of human peripheral blood lymphocytes. It is also suggested that these are two of the immune system-mediated mechanisms responsible for the indirect antitumor effect of IFN-beta in vivo.

[Study on human lymphoblastoid interferon-alpha (MOR-22): Part II. Anticellular effects].

The direct and indirect anti-cell proliferation effects of IFN-alpha produced by BALL-1 cells were examined. Of 23 cell lines, 7 including 2 normal diploids were resistant to a dose 1000 IU/ml of IFN-alpha. On the other hand, 16 cell lines were sensitive and showed IC50 values of less than 1000 IU/ml. This anti-cell proliferation spectrum showed no specificity to tumor origin and its activity was reversible. Cellular oncogene expression was markedly suppressed by IFN-alpha treatment. This finding showed that there might be some correlation between anti-cell proliferation activity and anti-oncogene expression activity. Furthermore, by activating natural killer cell activity and antibody dependent cell-mediated cytotoxicity, IFN-alpha potentiated indirect anti-cell activity.

[Study on human lymphoblastoid interferon-alpha (MOR-22): Part III. Antitumor effect on nude mouse-transplanted human tumors].

The effects of human lymphoblastoid interferon-alpha (MOR-22) on the growth of xenografted human tumors in nude mice were examined. IFN-alpha was administered subcutaneously to mice with renal tumor (ACHN), intratumorally for glioblastoma (U-373MG) or intravenously with uterine cervical tumor (HeLa S3). The dosages of IFN-alpha were 2 X 10(4)-5 X 10(5) IU/mouse for ACHN tumor, 1 X 10(5)-5 X 10(5) IU/mouse for U-373MG tumor and 3 X 10(4)-1 X 10(5) IU/mouse for HeLa S3 tumor. IFN-alpha inhibited the growth of these tumors in a dose-dependent manner.

Deferoxamine mesylate inhibits bacterial growth in vivo.

Iron is one of the essential element for bacterial growth. To study the effect of chelation of iron on bacterial growth, 17 different strains of bacteria were cultured in chocolate-agar medium in the presence or absence of deferoxamine methylate (DM), a potent chelating agent for iron. A fairly low concentration of DM (10 mg/ml) in the culture medium markedly inhibited the growth rates of the three bacterial strains, B. catarrhalis, N. meningiditis and N. gonorrhoeae. In vivo effect of DM was also tested in mice that were intraperitoneally inoculated with a lethal number of N. meningiditis (2 x 10(8)/animal). After 40 h of inoculation, more than 90% of the control animals died for acute peritonitis. In contrast, animals which were intraperitoneally administered with DM showed a marked resistance to the inoculated bacteria without showing toxic effects of the agent: less than 30% of animals died of acute peritonitis caused by infection within 40 h. The results suggest that, in addition to the currently used antibiotics. DM might also be an useful therapeutic agent for treatment of bacterial infection.

In vivo anti-tumor efficacy of polyethylene glycol-modified tumor necrosis factor-alpha against tumor necrosis factor-resistant tumors.

We previously reported that the optimally PEGylated tumor necrosis factor-alpha (MPEG-TNF-alpha), in which 56% of the TNF-alpha-lysine amino groups were coupled with polyethylene glycol (PEG), had about 100-fold greater anti-tumor effect than native TNF-alpha. Here, we assessed the usefulness of MPEG-TNF-alpha as a systemic anti-tumor therapeutic drug, using B16-BL6 melanoma and colon-26 adenocarcinoma, which have been reported to be resistant to TNF-alpha in vivo, as compared with Meth-A fibrosarcoma. MPEG-TNF-alpha markedly inhibited the growth of both tumors without causing any TNF-alpha-mediated side-effects, whereas native TNF-alpha had no anti-tumor effects and caused adverse side-effects. In addition, MPEG-TNF-alpha drastically inhibited the metastatic colony formation of B16-BL6 melanoma. MPEG-TNF-alpha may, thus, be a potential systemic anti-tumor therapeutic agent.

Molecular design of conjugated tumor necrosis factor-alpha: synthesis and characteristics of polyvinyl pyrrolidone modified tumor necrosis factor-alpha.

We conjugated tumor necrosis factor-alpha (TNF-alpha) with the synthetic polymeric modifier polyvinyl pyrrolidone (PVP) to facilitate its clinical use for anti-tumor therapy. TNF-alpha was chemically conjugated with the terminal carboxyl-bearing PVP at one end of its main chain, which was radically polymerized via the formation of an amide bond between the lysine amino groups of TNF-alpha and carboxyl group of PVP. In vitro specific bioactivity of PVP-conjugated TNF-alpha (PVP-TNF-alpha) relative to that of native TNF-alpha gradually decreased with increases in the degree of PVP attachment. In contrast, PVP-TNF-alpha in which 40% of TNF-alpha lysine residues were coupled with PVP (MPVP-TNF-alpha) exhibited the highest anti-tumor activity among the conjugated derivatives examined. MPVP-TNF-alpha had more than 200-fold higher anti-tumor efficacy than native TNF-alpha, and the anti-tumor activity of MPVP- TNF-alpha was more than 5-fold stronger than that MPEG- TNF-alpha which had the highest anti-tumor activity among PEG-conjugated TNF-alphas examined. Additionally, a high dose of native TNF-alpha induced toxic side-effects such as body weight reduction, piloerection and tissue inflammation, while no side effects were observed following i.v. administration of MPVP-TNF-alpha. The plasma half-life of MPVP-TNF-alpha (360 min) was about 80 and 3-fold longer than those of native TNF-alpha (4.6 min) and MPEG-TNF-alpha (122 min), respectively. These results suggested that PVP is a useful polymeric modifier for increasing the anti-tumor activity of PVP.

In vivo antitumor mechanism of natural human tumor necrosis factor involving a T cell-mediated immunological route.

We have investigated the in vivo antitumor mechanism of natural human tumor necrosis factor (n-TNF) isolated from a culture of human leukemic B cell line (BALL-1), especially its action as an immunomodulator, and found that the in vivo antitumor effect of n-TNF on Meth A sarcoma implanted in BALB/c mice pretreated with monoclonal antibody against T cell-specific surface antigen (Thy-1) was significantly diminished. Furthermore, when BALB/c mice were treated with T cell subset-specific monoclonal antibodies, anti-L3T4 or anti-Lyt-2.2, the antitumor effect of n-TNF on Meth A sarcoma was significantly reduced. Therefore, it was suggested that the in vivo antitumor mechanism of n-TNF might involve a T cell-mediated immunological route.

The inhibition of neoplastic cell proliferation with human natural tumor necrosis factor.

Purified human natural tumor necrosis factor (n-TNF) was prepared by stimulating human leukemic B cell line (BALL-1) with Sendai virus. The colony formations of all of 18 human cancer-derived abnormal cell lines were suppressed by 10(1)-10(6) U/ml of n-TNF, while n-TNF was nontoxic to all human normal fibroblast cells. This in vitro inhibition of cell growth was reversible. In breast adenocarcinoma MCF7 cells treated with n-TNF a specific decrease of DNA synthesis was observed, and DNA histograms showed a block at G1 in the cell cycle. In vivo studies revealed that n-TNF suppressed the tumor growth of murine Meth A sarcoma, human renal adenocarcinoma (ACHN), malignant melanoma (SK-MEL-28) and glioblastoma (U-373 MG). Isobologram analysis showed that n-TNF synergistically inhibited cell growth in combination with human natural interferon (IFN)-a. In vivo synergism of n-TNF and IFN-a was also found in the U-373 MG tumor model implanted into nude mice.

Chemical modification of natural human tumor necrosis factor-alpha with polyethylene glycol increases its anti-tumor potency.

Natural human tumor necrosis factor-alpha (TNF-alpha) was chemically modified with an active ester of monomethoxy polyethylene glycol (PEG). The molecular weight of PEG-modified TNF-alpha depended on the reaction time as well as the initial molar ratio of PEG to TNF-alpha. The specific activity of modified TNF-alpha was gradually reduced with increase in the degree of PEG-modification, but the plasma half-life of TNF-alpha was increased by up to 40-fold. Modified TNF-alpha showed approximately 100 times greater anti-tumor potency than unmodified TNF-alpha. Covalent attachment of PEG to TNF-alpha thus increased the bioavailability of TNF-alpha, and may facilitate its potential therapeutic use.

Impaired fertility in female mice lacking urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI) is a serine proteinase inhibitor that is found in blood and urine. To investigate the physiological functions of UTI in vivo, we generated UTI-deficient mice by gene targeting. The mice showed no obvious abnormalities and appeared healthy. However, the females displayed a severe reduction in fertility. Wild-type embryos developed normally when transplanted into UTI-deficient female mice, suggesting that UTI-deficient females have a normal ability to maintain pregnancy. The number of naturally ovulated oocytes from UTI-deficient mice was greatly reduced compared with that from wild-type mice. Histologically, oocytes with disorganized corona radiata were frequently seen in the ovaries of UTI-deficient mice after hormonal stimulation. When ovaries from UTI-deficient mice were transplanted into wild-type mice, pups derived from the transplanted ovaries were obtained, suggesting that the ovary of UTI-deficient mice functions normally if UTI is supplied from the systemic circulation. These results demonstrate that UTI plays an important role in the formation of the stable cumulus-oocyte complex that is essential for oocyte maturation and ovulation.