Photoallergenicity of a fluoroquinolone antibacterial agent with a fluorine substituent at the 8-position in guinea pigs exposed to long-wavelength UV light.

The 8-position of the quinolone ring of balofloxacin (BLFX), one of fluoroquinolones, was replaced with fluorine to obtain the 8-F. When an aqueous solution of bovine serum albumin (BSA) containing the 8-F was exposed to long-wavelength UV light (UVA) at a rate of 2.5 J/cm2, the absorbance of BSA at 300 nm or longer wavelengths increased markedly in comparison to that of native BSA. In addition, when a homogenate of skin tissue from Hartley guinea pigs was exposed to UVA (2.5 J/cm2) in the presence of the 8-F and then injected subcutaneously into guinea pigs, the animals produced IgG class antibody specific to the 8-F and its UVA-irradiation product. No such phenomenon, however, was observed when the parent compound, i.e. , BLFX which possesses a methoxy group at the 8-position, was used instead of the 8-F. In a subsequent experiment, the 8-F was administered either orally or topically to the shaved neck of guinea pigs and then irradiated with UVA (5 J/cm2) once daily for 5 days. When the treated animals were challenged by a combination of UVA irradiation (5 J/cm2) and either an oral or intradermal administration of the 8-F, 2 and 3 of the 5 animals showed redness and erythema on the irradiated area, respectively. However, no change was observed when BLFX was used instead of the 8-F. These results suggest that the introduction of a fluorine substituent to the 8-position of quinoline ring of fluoroquinolones induces photoallergic responses in which the fluoroquinolone or its photo-denatured product(s) act as an allergen.

Reduced phototoxicity of a fluoroquinolone antibacterial agent with a methoxy group at the 8 position in mice irradiated with long-wavelength UV light.

A newly developed fluoroquinoline, Q-35 (8-OCH3), in which a methoxy group was substituted at the 8 position of the quinoline nucleus, was very stable under irradiation with long-wave UV light (UVA). Derivatives, a fluoroquinolone with no substitution (the 8-H analog) and one in which a fluorine was substituted (the 8-F analog), were degraded in their solutions by the UVA irradiation. The phototoxic inducibility by these derivatives was further studied in a murine model. When mice were dosed orally with 800 mg of Q-35 (8-OCH3) per kg of body weight, the maximum dose given, and exposed to the UVA light, no inflammatory lesions were observed in their ears. Ear redness was marked in mice given more than 12.5 mg of the 8-F analog or 200 mg of the 8-H analog per kg. Histopathological changes, edema, and infiltration of neutrophils were also observed microscopically in groups receiving the 8-H or 8-F analog but not in groups receiving Q-35 (8-OCH3). Similar inflammatory reactions were observed to occur in a dose-dependent manner with other available fluoroquinolone antibacterial agents such as lomefloxacin, enoxacin, norfloxacin, ciprofloxacin and ofloxacin. These results suggest that the introduction of a methoxy group at the 8 position of the quinolone nucleus is important for the reduction of phototoxicity.

Efficacy of oral administration of live herpes simplex virus type 1 as a vaccine.

Mice given herpes simplex virus type 1 (HSV-1) (Miyama +GC strain) intragastrically via a stainless-steel cannula were rendered immune to subsequent lethal intraperitoneal (i.p.) challenge with HSV-1. The orally administered HSV-1 was completely inactivated in the stomach within a few minutes of inoculation. However, systemic immunity was established 14 days after oral inoculation with the virus and retained for up to 6 months. The mechanisms of establishing systemic immunity were investigated by means of adoptive transfer comparisons. When splenic cells from HSV-1-immunized mice were transplanted into nonimmunized mice, all of the recipient mice survived after a lethal i.p. challenge with the virus. Immunity was not established in antithymocyte serum-treated mice or by transfer of serum from immunized to nonimmunized mice. In addition, all HSV-1-immunized mice died after lethal challenge with HSV-2 and influenza virus A. These findings suggest that the immunity was virus specific, with T lymphocytes playing a major role in its establishment. The present study therefore supports the possibility of oral immunization with live HSV-1 as a vaccine.

Ultrastructural characteristics of lymphokine-activated killer cells of the rat in comparison with natural killer cells.

Lymphokine-activated killer (LAK) cells with a broad spectrum of tumor cell killing have been reported to be related to natural killer (NK) cells morphologically and phenotypically. We here examine the ultrastructural characteristics of LAK cells of the rat, comparing them to those of normal and OK-432-activated NK cells. Results show that, five days after the culturing of spleen lymphocytes with human recombinant interleukin-2, there were induced LAK cells, which were large granular lymphocytes and had a cytotoxic capacity against NK-resistant P-815 tumor cells. They were larger in size than NK cells and richer in cell organelles such as ribosomes, rough endoplasmic reticulum, a Golgi apparatus, granules and vesicles. The granules of LAK cells were shown to be related to multivesicular bodies as those of NK cells; they included multivesicular bodies, fully dense granules and intermediate forms between them. The average numbers and sizes of the granules and the proportion of multivesicular bodies and intermediate forms among the total granules were greater in LAK cells than in NK cells. The density of the small vesicles packed in multivesicular bodies and intermediate forms was much higher in LAK cells. At the contacting surface of the LAK cells bound to the target cells, exocytosis of multivesicular bodies was shown to occur. We recognized here two populations of LAK cells with different types of vesicles, one containing rod-cored vesicles and the other a new type of vesicles termed "demilune-cored vesicles". The latter vesicles were the same in size as the rod-cored ones and contained a dense core located eccentrically. Between these two populations of LAK cells, there was no difference concerning the profile of the dense granules. The present study indicates that, although LAK and NK cells share several ultrastructural features, the former show markedly enriched cell organelles, which indicate an accelerated metabolism of the cell for continuous proliferation.

Production of interleukin 6 by human spleen cells stimulated with streptococcal preparation OK-432.

The streptococcal preparation OK-432 was tested for the ability to stimulate human spleen leukocytes (SPL) for generation of interleukin 6 (IL-6). When SPL were cultured with OK-432 for 24 h in serum-free T medium, the cell-free supernatant induced production of IgM in the SKW6.CL-4 and IgG in the CESS human B cell line, while no such activity was detected in unstimulated SPL culture. The activity was neutralized by treatment with antiserum directed against B cell stimulatory factor 2 (BSF-2). An optimum production of BSF-2 was observed when SPL were stimulated with 10 micrograms/ml of OK-432. The culture supernatant also induced proliferation of IL-6-dependent murine hybridoma MH-60.BSF2 (hybridoma growth factor; HGF). It is thus evident that the molecule produced by OK-432-activated human SPL is BSF-2/HGF/IL-6. These results indicate that the antitumor agent OK-432 stimulates human spleen cells to produce IL-6.

Effects of X-ray irradiation on natural killer (NK) cell system. I. Elevation of sensitivity of tumor cells and lytic function of NK cells.

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.

Effects of X-ray irradiation on natural killer (NK) cell system. II. Increased sensitivity to natural killer cytotoxic factor (NKCF).

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.

Therapeutic effect of OK-432 induced endogenous TNF on tumor bearing mice and cancer patients.

The therapeutic effect of OK-432 induced endogenous TNF on tumor bearing mice and cancer patients was investigated. OK-432 (10 KE/mouse) was administered intraperitoneally to Balb/c mice 7 days prior to the transplantation of Meth A cells (1 x 10(6)/mouse) into the abdominal cavity. And at day 1 of tumor inoculation, 1 KE/mouse of OK-432 was administered intraperitoneally. The significant prolongation of life span was observed in these mice. On the basis of these observation, therapeutic effect of endogenous TNF on cancer patients was clinically evaluated. OK-432 was administered intraperitoneally or intrapleurally to cancer patients with peritonitis carcinomatosa or pleuritis carcinomatosa 4 times (10KE each) every other day and 50KE of OK-432 was readministered with the interval of 7 days. An appreciable activity of TNF was detected in peritoneal fluids or pleural effusion, and the significant decreasing of these fluids was observed. It is therefore concluded that these therapeutic approach may well be taken into account in treatment of cancer.

Characterization of a cytotoxic factor induced in mouse peritoneal fluid by OK-432.

A cytotoxic factor (peritoneal cytotoxic factor, PCF) was strongly induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with OK-432. In order to clarify characteristics of PCF, physicochemical and immunological studies were conducted. When incubated with LPS, the macrophages from mice primed with OK-432 induced PCF whereas the lymphocytes did not. These results indicate that PCF is different from lymphotoxin. PCF appears to be quite similar to tumor necrosis factor (TNF) in the serum for the following reasons: The two factors are similar in the mode of cytotoxic action in vitro; both factors have a tumor necrotizing effect when injected into tumor bearing mice; both are produced from macrophages; they are similar in physicochemical characteristics; and the cytotoxic activity of PCF is totally abolished by anti-TNF serum.

[Induction of a cytotoxic factor into the peritoneal fluid (PCF) by OK-432. II. Physicochemical and immunological characteristics of PCF].

A cytotoxic factor (PCF = peritoneal cytotoxic factor) was strongly induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with OK-432. The similarity in biological activity of PCF, TNF and lymphotoxin led us to study the relationships among the three. When incubated with LPS, the macrophages from the mice primed with OK-432 induced PCF, whereas the lymphocytes did not. These results indicate that PCF is different from lymphotoxin. PCF appears to be identical to Tumor Necrosis Factor (TNF) in the serum for the following reasons: The two factors are similar in their modes of cytotoxic action in vitro. Both factors have a tumor-necrotizing effect when injected into tumor-bearing mice. Both are produced from macrophages. They are similar in their physicochemical characteristics. The cytotoxic activity of PCF was totally abolished by anti-TNF serum.

[Induction of mouse peritoneal cytotoxic factor by OK-432. (3). Comparison between LPS and OK-432 as eliciting agents].

By injection of OK-432, a cytotoxic factor was induced in peritoneal fluids of mice which had been primed with OK-432. Two-step stimulation (priming and eliciting) was always necessary to induce the cytotoxic factor. OK-432-primed mice did not produce soluble cytotoxic factor spontaneously and no cytotoxic activity was detected in the mice treated by a single injection of OK-432 as an eliciting agent. High doses of OK-432 were required to prime mice for the production of cytotoxic factor, whereas a small amount was enough to elicit it. Pathological studies were also conducted in order to clarify whether the mice were safe under the conditions in which PCF had been induced. Moderate liver damage was observed in the mice injected with OK-432 and LPS, whereas no histological change in the liver or spleen was observed in the mice treated with OK-432 alone. These results suggest that OK-432 is a good candidate as an inducer of cytotoxic factor in the peritoneal cavity.

[Production of a cytotoxic factor in mouse peritoneal fluid by OK-432, a streptococcal preparation].

A cytotoxic factor was induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with a streptococcal antitumor preparation, OK-432. No cytotoxic effect on L-929 cells was observed in the peritoneal fluids of mice singly treated with OK-432 or LPS. Various mouse and human tumor cell lines were effectively killed by this peritoneal cytotoxic factor, though normal cell lines were insensitive, which indicates that this factor is not species-specific. The highest level of cytotoxic activity was obtained when LPS was given to mice 5 days after the injection of OK-432. The optimal time for collection of peritoneal fluids for the cytotoxic factor was 2h following the LPS injection. Interferon activity was found to be negative by the plaque reduction test using L-929 cells with vesicular stomatitis virus.

Release of tumor necrosis factor (TNF) into mouse peritoneal fluids by OK-432, a streptococcal preparation.

A cytotoxic factor was induced in peritoneal fluid by injection of OK-432 in mice which had been primed with OK-432. Two-step stimulation (priming and eliciting) was always necessary to induce the cytotoxic factor. OK-432-primed mice did not produce soluble cytotoxic factor spontaneously and no cytotoxic activity was detected in the mice treated by a single injection of OK-432 as an eliciting agent. This observation was also confirmed by in vitro experiments. Only when activated macrophages were incubated with OK-432 (or with lipopolysaccharide) was cytotoxin released into medium supernatant. High doses of OK-432 were required to prime mice for the production of cytotoxic factor, whereas a small amount was enough to elicit. The peritoneal cytotoxic factor obtained by OK-432 injection appears to be identical to tumor necrosis factor in the serum for the following reasons: The two factors are similar in mode of cytotoxic action. Both are produced from macrophages. They are similar in physicochemical characteristics. The cytotoxicity of the peritoneal cytotoxic factor was totally abolished by anti-TNF serum.

Therapeutic effect of endogenous tumor necrosis factor on ascites Meth A sarcoma.

The therapeutic effect of endogenous tumor necrosis factor (TNF) on Meth A ascites fibrosarcoma in mice was investigated. Serum and peritoneal fluid from tumor bearing mice treated with OK-432 and LPS were cytotoxic to tumor cells in vitro. The peak of cytotoxicity in both the serum and peritoneal fluid was found in the fraction corresponding to a molecular weight of approximately 54,000-56,000 on HPLC and the pI was found to be 4.9-5.1 by isoelectric focusing. These results are consistent with previously reported findings on TNF, and indicate that endogenous TNF has a satisfactory life-prolonging effect. The tumor necrosis factor (TNF) is considered to be one of the clinically most promising anti-cancer cytokines because of its potent and very specific antitumor effect on target cells (Carswell, Old, Kassel, Green, Fiore & Williamson, 1975; Matthews & Watkins, 1978; Niitsu, Watanabe & Urushizaki, 1984). TNF as an anti-cancer cytokine for the treatment of cancer may be applied in one of the two following ways: by administration of purified TNF or by endogenously inducing TNF in cancer bearing individuals. The antitumor effects of TNF administered exogenously have been examined using crude preparations or serum containing TNF (tumor necrosis serum, TNS) (Carswell et al., 1975; Watanabe, Niitsu, Sone, Neda, Ishigaki & Urushizaki, 1984). In a previous paper we reported that mice primed with OK-432 and challenged with endotoxin produced a soluble cytotoxic factor in peritoneal fluids (Yamamoto, Nagamuta, Usami, Sugawara, Watanabe, Niitsu & Urushizaki, 1985; Nagamuta, Yamamoto, Usami, Sugawara, Watanabe, Niitsu & Urushizaki, 1985). Ths peritoneal cytotoxic factor (PCF) had cytostatic and/or cytotoxic effect not only on mouse tumor cell lines but also on human tumor cell lines without species specificity. Normal cell lines were not affected. Here we report the endogenous production of TNF in tumor bearing mice and its antitumor effects.

Production of cytotoxic factor into mouse peritoneal fluid by OK-432, a streptococcal preparation.

A cytotoxic factor was induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with a streptococcal antitumor preparation, OK-432. No cytotoxic effect on L-929 cells was observed in the peritoneal fluids of mice singly treated with OK-432 or LPS. Various mouse and human tumor cell lines were effectively killed by this peritoneal cytotoxic factor, though normal cell lines were insensitive, which indicates that this factor is not species-specific. The highest level of cytotoxic activity was obtained when LPS was given to mice 5 days after the injection of OK-432. The optimal time for collection of peritoneal fluids for the cytotoxic factor was 2 h following the LPS injection. Interferon activity was found to be negative by the plaque reduction test using L-929 cells with vesicular stomatitis virus. These results suggest that this cytotoxic factor is similar to the tumor necrosis factor (TNF) in the mouse serum.