Efficacy of calcium-EDTA as an inhibitor for metallo-ß-lactamase in a mouse model of Pseudomonas aeruginosa pneumonia.

In this study, we have evaluated the efficacy of calcium-EDTA (Ca-EDTA) as an inhibitor of bacterial metalloenzymes, such as metallo-ß-lactamase (MBL) and other proteases, in a mouse model of Pseudomonas aeruginosa pneumonia. The simultaneous presence of Ca-EDTA (32 µg/ml) reduced the MICs of imipenem (IPM) in all MBL-producing P. aeruginosa isolates (IMP-1, -2, -7, and -10 and VIM-2) but not non-MBL-producing strains. In the pneumonia model, mice were intranasally infected with MBL-producing P. aeruginosa and then kept under conditions of hyperoxia to mimic ventilator-associated pneumonia. With both intranasal and subcutaneous administrations, Ca-EDTA significantly potentiated survival benefits of IPM compared to those of IPM alone. Ca-EDTA combination therapy induced a significant reduction of the bacterial burden in the lungs (P < 0.05). Furthermore, the inhibition activity of Ca-EDTA against MBL activity was confirmed by using the purified IMP-1 enzyme, which was characterized by a 50% inhibitory concentration (IC(50)) of 55 ± 8.2 µM. Finally, the protective effects of Ca-EDTA were demonstrated by culture supernatant-induced epithelial cell damage and acute lung injury in mice. These data suggest the therapeutic potential of Ca-EDTA not only by the blocking of MBLs but also by neutralizing tissue-damaging metalloproteases in P. aeruginosa infections.

Efficacy of colistin combination therapy in a mouse model of pneumonia caused by multidrug-resistant Pseudomonas aeruginosa.

OBJECTIVES: Multidrug-resistant Pseudomonas aeruginosa (MDRP) is becoming a serious problem in hospitals, especially in patients on ventilators. Recent data demonstrate that colistin may be effective for these patients, although limited in vitro and in vivo data are available. Our aim was to identify further characteristics of colistin for the therapy of pneumonia caused by MDRP. METHODS: The effects of colistin on clinical strains of MDRP were examined by susceptibility test, time-kill assay, lipopolysaccharide (LPS)-blocking assay and a mouse pneumonia model, alone or in combination with other antibiotics. For the pneumonia model, mice were intranasally infected with bacteria and kept in hyperoxic conditions to mimic ventilator-associated pneumonia. RESULTS: As a single agent, colistin exhibited the strongest activity of the antimicrobial agents tested. In combination, maximum synergy was observed with colistin plus rifampicin. As expected, co-incubation of bacterial culture supernatants with colistin significantly reduced LPS activities with an associated decrease in cellular cytotoxicity. In the pneumonia model, intranasal, but not intravenous, colistin combined with rifampicin produced maximum survival protection. Pharmacokinetic analysis of colistin demonstrated the superiority of intranasal administration, judging from the compartmentalized high concentration and the long half-life in the lungs. Moreover, colistin therapy significantly decreased both production of inflammatory cytokines and LPS activity, even at a dose effecting no change in the bacterial burden in the lung. CONCLUSIONS: These data strongly suggest that colistin may be an important option for combination therapy against critical MDRP infections. For pneumonia especially, intranasal colistin with rifampicin may be beneficial not only for synergistic antibacterial activity, but also for blocking LPS.

Preventive effect of hochu-ekki-to, a Japanese herbal medicine, on bleomycin-induced lung injury in mice.

OBJECTIVE: Pulmonary fibrosis is thought to be closely associated with the T-helper type-2 (Th2) immune response. Recent studies have shown that hochu-ekki-to (TJ-41), a Japanese herbal medicine, may correct the Th1/Th2 imbalance skewed to Th2. The present study was designed to investigate the preventive effect of TJ-41 on the development of bleomycin (BLM)-induced lung injury in mice. METHODS: Female C57BL/6 mice were divided into a group given ordinary feed and another group given the same feed plus TJ-41 mixed in at a dose of 1 g/kg/day. Both groups were maintained on this diet for 8 weeks before and 5 weeks after administration of 2 mg/kg BLM intratracheally. RESULTS: Mortality after BLM-induced lung injury was significantly lower in the TJ-41-treated mice. The hydroxyproline content and fluid content in the lung on day 35 was significantly lower in the TJ-41-treated mice. Histologically, TJ-41 reduced the number of infiltrating cells, thus ameliorating the destruction of the lung architecture, and attenuated the lung fibrosis score. Furthermore, TJ-41 inhibited the expression of the interleukin-5/interferon-gamma mRNA ratio in the lung on day 7. CONCLUSIONS: Treatment with TJ-41 partially prevented experimental lung fibrosis through the correction of the Th1/Th2 imbalance skewed to Th2.

Preventive effect of Hochu-ekki-to on lipopolysaccharide-induced acute lung injury in BALB/c mice.

This study was designed to investigate the effect of Hochu-ekki-to (TJ-41), a Japanese herbal medicine, on the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. ALI was induced in female BALB/c mice by the intranasal administration of 0.1 mg/kg LPS. The mice were divided into a group receiving normal feed and another group receiving feed mixed with TJ-41 at a dose of 1 g/kg/day for 8 weeks before LPS challenge. In the bronchoalveolar lavage fluid, the preadministration of TJ-41 caused significant reduction in the absolute number of total cells, neutrophils, and macrophages. The preadministration of TJ-41 significantly inhibited increases in the serum level of keratinocyte chemoattractant (KC), which is a murine chemotaxin for neutrophils that corresponds to human interleukin-8, with respect to its concentration at 24 h after LPS challenge. Furthermore, the histopathologic findings indicated that alveolitis with leukocyte infiltration in the alveolar space was less severe in the TJ-41-treated mice than in the control mice. These findings indicated that the preadministration of TJ-41 could show an inhibitory effect on ALI in this experimental murine system associated with the suppression of chemokine production.

Inhibitory effect of antimicrobial agents and anisodamine on the staphylococcal superantigenic toxin-induced overproduction of proinflammatory cytokines by human peripheral blood mononuclear cells.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), produces superantigenictoxins, such as toxic shock syndrome toxin-1 (TSST-1). TSST-1 abnormally activates T cells to overproduce inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) leading to shock. In this study, we investigated the inhibitory effect of antimicrobial agents and anisodamine (a Chinese herbal extract) on TSST-1-induced cytokine production. Among the macrolides and related agents examined, azithromycin and rokitamycin showed the greatest inhibitory activity against the TSST-1-induced cytokine production. This inhibitory effect was similar to that of anisodamine, which, however, had no inhibitory activity against bacterial growth. Vancomycin, teicoplanin, arbekacin, and linezolid (anti-MRSA and related agents) had no significant inhibitory effect on cytokine production. The inhibitory effect of the drugs on cell proliferation was not significant. These data indicate that some antimicrobial agents, e.g., azithromycin and rokitamycin, manifest anti-superantigenic toxin activity through the inhibition of cytokine production, just like anisodamine.

Transferrin saturation versus reticulocyte hemoglobin content for iron deficiency in Japanese hemodialysis patients.

BACKGROUND: Iron deficiency is a frequent cause of recombinant human erythropoietin (rhEPO)-resistant anemia in hemodialysis patients. Both reticulocyte hemoglobin content (CHr) and transferrin saturation (TSAT) have been proposed as markers of iron deficiency, but it is unclear which parameter is superior. METHODS: To compare the efficacy of CHr and TSAT as an indicator for treatment of iron deficiency, we conducted a single-center, open-label, prospective, randomized, controlled trial at the Kidney Center in Shinraku-en Hospital of 197 Japanese patients on chronic hemodialysis. After 4 weeks of run-in period during which iron supplementation was suspended, 100 patients who were randomized to the CHr group received 240 mg iron colloid intravenously over 2 weeks when CHr less than 32.5 pg, and 97 patients who were randomized to the TSAT group received the same doses of iron colloid when TSAT less than 20%. We measured the rhEPO dose needed to maintain prestudy hematocrit levels, hematocrit, CHr, TSAT, serum ferritin, percentage of hypochromic red blood cells, and total iron administered. RESULTS: Sixteen weeks later, 94 patients in the CHr group and 89 patients in the TSAT group finished the study. The doses of rhEPO required decreased by 35.8% (4081 to 2629 U/week, P < 0.005) in the TSAT group, but not significantly in the CHr group (4121 to 3606 U/week). Although CHr increased promptly after the iron administration in both groups, TSAT increased only in the TSAT group. CONCLUSIONS: Although CHr reflects the iron status more sensitively, TSAT is a better clinical marker for iron supplementation therapy.

[A case of interstitial pneumonia exacerbated by kampo-induced pneumonitis].

A 65-year-old woman who had complained of non-productive cough since May 1998 visited our hospital on November 5, 2000. She had been treated at another hospital with Kampo (Chinese herbal medicine), including Moku-boui-to, Bakumon-do-to, and Saiko-keishi-kankyo-to for chronic non-productive cough. Chest radiographs and CT films showed the reticular shadows that had been present in 1998, in both lower lung fields, and also demonstrated new reticular shadows in the right upper lung field and left lingular segment. Laboratory data revealed hypoxemia and pulmonary function tests revealed restrictive ventilatory disturbance, so she was admitted to our hospital on November 9, 2000. After the cessation of Kampo treatment, her symptoms disappeared, and the hypoxemia, restrictive disturbance, and reticular shadows in the chest radiograph gradually improved. Video-assisted lung biopsy specimens showed thickened alveolar walls with lymphocyte and eosinophil infiltration. A leukocyte migration test was positive for Moku-boui-to, Bakumon-do-to, and weakly positive for Saiko-keishi-kankyo-to. Although no challenge test for Kampo was performed, we diagnosed this case as interstitial pneumonia exacerbated Kampo-induced pneumonitis based on these clinical, laboratory and histological findings.

Liver injury due to sequential activation of natural killer cells and natural killer T cells by carrageenan.

BACKGROUND/AIMS: Carrageenan is a high molecular weight polysaccharide and is widely used as a food additive for the solidification of plant oils and the thickening of many beverages. It is known that acute toxicity of carrageenan is possibly induced by the activation of phagocytic cells. We investigated other effects of carrageenan on lymphocytes in this study. METHODS: Carrageenan was intraperitoneally injected once into mice and phenotypic and functional characterizations were conducted in various immune organs. RESULTS: Natural killer (NK) cells were prominently activated in the liver, lungs, and spleen. A time-kinetic study showed sequential activation of NK and natural killer T (NKT) cells in the liver on days 3-10 after the injection. In parallel with the activation of NK and NKT cells in number, NK and NKT cytotoxicities were augmented. At this time, liver injury was induced, accompanied by massive hepatic necrosis and the elevation of transaminases. The in vivo elimination of NK cells reduced the liver injury induced by carrageenan. Direct binding of carrageenan onto NK cells was also demonstrated. Such a binding then induced a subsequent production of IFN gamma. Perforin molecules of NK cells were responsible for this liver injury. CONCLUSIONS: These results suggest that not only phagocytic cells but also primitive lymphocyte (mainly NK cells) subsets might be important targets for the acute toxicity of carrageenan.