Fuminari Sonoda

Therapeutic effects of a human antiflagella monoclonal antibody in a neutropenic murine model of Pseudomonas aeruginosa pneumonia.

Human immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) reactive with type-specific Pseudomonas aeruginosa lipopolysaccharide (LPS) and flagella were compared for their protective activities against Fisher immunotype 2 P. aeruginosa pneumonia in neutropenic mice. The activity of the antiflagella MAb at a dose of 500 micrograms per mouse was comparable to that of the anti-LPS MAb at the same dose. In vivo protection was correlated with bacterial density in the lung tissue and blood of infected mice. In vitro data suggested that the protective activity of the antiflagella MAb was due more to inhibition of bacterial motility than to opsonophagocytosis of bacteria by alveolar macrophages. In contrast, the protective activity of the anti-LPS MAb was primarily related to alveolar macrophage-mediated opsonophagocytosis. Antiflagella MAb at a dose of 500 micrograms combined with oral sparfloxacin at a subtherapeutic dose of 62.5 micrograms produced a significant increase in survival (P < 0.05) compared with that produced by either agent alone or no treatment. The additive effects between the antiflagella MAb and sparfloxacin at sub-MICs on the inhibitory effects of bacterial motility supported the in vivo effect of the combination. These data suggest that human isotype-matched antiflagella and anti-LPS MAbs have similar protective activities against Pseudomonas pneumonia in neutropenic mice, despite discrete mechanisms of antibody-matched protection. In addition, in vivo synergy was demonstrated between antiflagella MAb and sparfloxacin in this model.

Airway Interleukin‐8 in Elderly Patients with Bacterial Lower Respiratory Tract Infections

We investigated the interleukin‐8 (IL‐8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents. The IL‐8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens. We also demonstrated that human recombinant IL‐8 at a range of 6.25‐25 ng/ml significantly enhanced opsonophagocytic killing of P. aeruginosa immunotype‐1 strain by human neutrophils in the presence of a serotype‐specific anti‐lipopolysaccharide monoclonal antibody and fresh normal human serum. These data suggest that the level of IL‐8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL‐8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.

Effects of the combination of lipopolysaccharide-specific monoclonal antibodies and sparfloxacin against Pseudomonas aeruginosa pneumonia in neutropenic mice.

The effects of the combination of a murine monoclonal antibody (MAb) specific for the O side chain of Pseudomonas aeruginosa Fisher immunotype 1 lipopolysaccharide and sparfloxacin in a neutropenic mouse model of P. aeruginosa pneumonia were examined. Under the condition that neither MAb at a dose of 500 micrograms per mouse administered intravenously nor a suboptimal dose of oral sparfloxacin (5 mg/kg of body weight) protected mice from challenge with a fatal dose, the combination therapy with MAb and sparfloxacin caused a significant increase in the survival rate (P less than 0.001 compared with either treatment alone). The effect of the combination was closely correlated to bacterial killing in plasma and lung tissue of infected mice. In vitro, a significant MAb-dependent, complement-mediated killing of P. aeruginosa was documented in the presence of sparfloxacin at one-half the MIC, while the killing was not observed in the absence of sparfloxacin. These in vivo and in vitro data suggest the usefulness of combination therapy with a lipopolysaccharide-reactive immunoglobulin G MAb and sparfloxacin in neutropenic patients with P. aeruginosa pneumonia.

Endogenous Tumor Necrosis Factor (TNF) α Mediates Neutrophil Accumulation at the Mid-Phase of a Murine Model of Pseudomonas aeruginosa Pneumonia

To determine the role of endogenous tumor necrosis factor (TNF) α on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF α activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa‐infected mice. In the case of fatal pneumonia, the TNF α activity in the BALFs appeared within 3 hr, peaked at 6–12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF α activity was detected in the plasma. The elevation of TNF α activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF α activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6–12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF α activity in the BALFs with anti‐murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0–49.7% at 6 hr and 49.3–54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti‐murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre‐immune serum). These data suggest that intra‐alveolar TNF α plays an important role in causing lung neutrophil accumulation at the mid‐phase of murine P. aeruginosa pneumonia.

Pharmacodynamic and Protective Properties of a Murine Lipopolysaccharide-Specific Monoclonal Antibody in Experimental Pseudomonas aeruginosa Pneumonia in Mice

We employed a Pseudomonas aeruginosa mouse pneumonia model to evaluate the ability of a murine monoclonal antibody (MAb) specific for the O‐side chain of P. aeruginosa Fisher Immunotype‐1 lipopolysaccharide (LPS) to achieve and sustain therapeutic levels in plasma and lung tissue, reduce bacterial populations in the lung, and prevent pneumonia‐associated mortality. An IgG3 MAb (Y1–5A4) administered to mice i.v. over a dose range of 125–1,000 μg/mouse produced plasma and lung tissue levels at 2 hr of 61–507 μg/ml and 4.3–150 μg/g, respectively. The 1,000 μg MAb dose reduced bacterial counts in lung tissue (logio cfu/g±S.D.) and blood (log10cfu/ml+S.D.) 20 hr post‐treatment (18 hr post‐challenge) from 10.00+ 0.66 to 7.66±0.91 (P<0.01) and from 4.39±0.81 to <3.0, respectively. Administration of MAb to mice in doses of 125–500 μg 2 hr prior to a 3 × 50% lethal bacterial challenge produced significant protection against death, with a calculated 50% protective dose of 167 μg. Protection was noted following administration of 1,000 μg of MAb up to 6 hr after bacterial challenge (P<0.05, compared with untreated control). Histological examination of lung tissue from infected mice revealed less acute inflammation, necrosis, and hemorrhage in MAb‐treated compared with untreated control animals and greater localization of Pseudomonas antigen within the phagocytic cells in alveolar space. These findings document the in vivo therapeutic efficacy of an LPS‐specific IgG MAb in a murine model of acute P. aeruginosa pneumonia, based in part upon the achievability of effective MAb concentrations in plasma and lung tissue.