No abstract

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy. Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation. This SUPER TWIG specifically bound to Stx with high affinity (Kd ‫؍‬ 1.1 ؋ 10 ؊6 M) and inhibited the incorporation of the toxin into target cells. Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx. Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E. coli O157:H7, even when administered after the establishment of the infection. The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced. Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E. coli.

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 causes bloody diarrhea and hemorrhagic colitis in humans, sometimes resulting in fatal systemic complications. Among the known Stx family members, Stx2 is responsible for the most severe forms of disease. Stx2 binds to target cells via multivalent interactions between its B-subunit pentamer and globotriaosyl ceramide. After binding, it is first retrogradely transported to the Golgi and then to the endoplasmic reticulum (ER). Using a multivalent peptide library approach, we identified a tetravalent peptide that exhibits a high affinity for the Stx2 B-subunit pentamer (KD = 0.13 microM) and markedly inhibits Stx2 cytotoxicity. The tetravalent peptide exerted its inhibitory effects by inducing aberrant cellular transport of Stx2. Although the tetravalent peptide/Stx2 complex was incorporated into cells and translocated to the Golgi, this process was followed by the effective degradation of Stx2 in an acidic compartment rather than by its transfer to the ER. This peptide thoroughly protected mice from a fatal dose of E. coli O157:H7 even when administered after an established infection. Thus, the multivalent peptide library approach enabled the identification of a peptide-based Stx2 inhibitor that has remarkable therapeutic potency and appears to function by inducing aberrant cellular transport and degradation of Stx2.

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

Although macrophages play a central role in the pathogenesis of septic shock, NK1+ cells have also been implicated. NK1+ cells comprise two major populations, namely NK cells and Vα14+NKT cells. To assess the relative contributions of these NK1+ cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of β2-microglobulin (β2m)−/− mice that are devoid of Vα14+NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that β2m−/− mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-γ following LPS challenge were significantly higher in β2m−/− mice, and endogenous IFN-γ neutralization or in vivo depletion of NK1+ cells rescued β2m−/− mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-γ producers. The Jα281−/− mice that are exclusively devoid of Vα14+NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of Vα14+NKT cells and IFN-γ from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely.

LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1−/− mice are far more resistant to Listeria monocytogenes infection than LFA-1+/− mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1−/− mice than in LFA-1+/− mice, 2) increased antilisterial resistance in LFA-1−/− mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1−/− mice than in LFA-1+/− mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1−/− mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1−/− mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.

We carried out a randomized study of 49 consecutive patients with unresectable primary lung cancer to determine whether clarithromycin (CAM), a 14-membered ring macrolide, can improve outcome. A total of 49 patients (42 patients with non-small-cell lung cancer and 7 patients with small-cell lung cancer) had received prior chemotherapy, radiotherapy or both during their hospital stay. They were randomly allocated into two study groups on the first visit after discharge: 25 patients (22 patients with non-small-cell lung cancer, 3 patients with small-cell lung cancer) were assigned to receive CAM (400 mg/day, orally), and 24 patiens (20 patients with non-small-cell lung cancer, 4 patients with small-cell lung cancer) did not receive CAM. CAM treatment after randomization was open and the treatment was to be continued as long as the patients could tolerate CAM. There was no significant difference in the median survival time for small-cell lung cancer between the CAM group and the non-CAM group. However, CAM treatment significantly increased the median survival time for non-small-cell lung cancer patients, the median survival for the CAM group was 535 days and that for the non-CAM group was 277 days. Analyses of prognostic factors showed that only treatment with CAM was predictive of longer survival for non-small-cell lung cancer, and other tested covariates had no effects on the prognosis. There were no remarkable side effects observed in the CAM group throughout treatment. We conclude that long-term treatment using CAM is beneficial for unresectable non-small-cell lung cancer patiens and that it can increase the median survival of patients with advanced disease.

The effects of erythromycin stearate over a concentration range of 0.1-10 mg/l on production of elastase, protease and leucocidin by clinical isolates of Pseudomonas aeruginosa were investigated. Growth of P. aeruginosa N42 in broth was not affected significantly during 24 h culture with erythromycin (0.1-10 mg/l), although extracellular protein contents were reduced by erythromycin at concentrations of 0.1-1.0 mg/l. Production of elastase and protease by strain N42 was significantly suppressed by erythromycin with a maximum inhibition at 0.5 mg/l, but the complete inhibition of enzyme production was not achieved. In contrast, leucocidin production by strain N42 was completely impaired by erythromycin at concentrations of 0.1-5.0 mg/l. Although the leucotoxic activity, as determined by vital staining, was not detected, the leucocidin fraction prepared from the autolysate of strain N42 cultured with 10 mg/l of erythromycin induced morphological changes in human leucocytes, resulting in release of elastase. Erythromycin exerted similar effects on other clinical isolates of P. aeruginosa. These findings indicate that erythromycin might have a role in P. aeruginosa infection, although it has no direct antibacterial activity.