Pseudomonas aeruginosa serotypes in nosocomial pneumonia: prevalence and clinical outcomes.

INTRODUCTION: Pseudomonas aeruginosa frequently causes nosocomial pneumonia and is associated with poor outcome. The purpose of this study was to assess the prevalence and clinical outcome of nosocomial pneumonia caused by serotype-specific P. aeruginosa in critically ill patients under appropriate antimicrobial therapy management. METHODS: A retrospective, non-interventional epidemiological multicenter cohort study involving 143 patients with confirmed nosocomial pneumonia caused by P. aeruginosa. Patients were analyzed for a period of 30 days from time of nosocomial pneumonia onset. Fourteen patients fulfilling the same criteria from a phase IIa studyconducted at the same time/centers were included in the prevalence calculations but not in the clinical outcome analysis. RESULTS: The prevalence of serotypes was: O6 (29%), O11 (23%), O10 (10%), O2 (9%), and O1 (8%). Serotypes with a prevalence of less than 5% were found in 13% of patients, 8% were classified as not typeable. Across all serotypes, 19% mortality, 70% clinical resolution, 11% clinical continuation, and 5% clinical recurrence were recorded. Age and higher APACHE II (Acute Physiology and Chronic Health Evaluation II) were predictive risk factors associated with probability of death and lower clinical resolution for P. aeruginosa nosocomial pneumonia. Mortality tends to be higher with O1 (40%) and lower with O2 (0%); clinical resolution tends to be better with O2 (82%) compared to other serotypes. Persisting pneumonia with O6 and O11 was, respectively, 8% and 21%; clinical resolution with O6 and O11 was, respectively, 75% and 57%. CONCLUSIONS: In P. aeruginosa nosocomial pneumonia, the most prevalent serotypes were O6 and O11. Further studies including larger group sizes are needed to correlate clinical outcome with virulence factors of P. aeruginosa in patients with nosocomial pneumonia caused by various serotypes; and to compare O6 and O11, the two serotypes most frequently encountered in critically ill patients.

Development of a 4-valent genotyping assay for direct identification of the most frequent Pseudomonas aeruginosa serotypes from respiratory specimens of pneumonia patients.

Pseudomonas aeruginosa is a common cause of nosocomial infections and is associated with high rates of mortality. In order to facilitate rapid and sensitive identification of the most prevalent serotypes of P. aeruginosa, we have developed a 4-valent real-time PCR-based assay using oligonucleotides specific for open-reading frames constituting the O-antigen-specific lipopolysaccharide loci of P. aeruginosa. The assay simultaneously detects and differentiates between each of the four serotypes IATS-O1, -O6, -O11 and serogroup 2 (IATS-O2, -O5, and -O16) with high sensitivity and specificity in a single-tube reaction. No cross-reactivity was observed with other serotypes of P. aeruginosa or other microbial species, and reproducibility was demonstrated regardless of template, i.e. purified DNA, bacterial culture and clinical specimens (broncho-alveolar lavage). The limit of detection of the assay was approximately 100 copies per reaction for IATS-O1, -O2 and -O11, and 50 copies per reaction for IATS-O6. Comparison of the assay specificity with a commercially available slide agglutination kit showed consistent results; however, the number of non-typable isolates was reduced by 15 % using the genotyping assay. Use of the 4-valent genotyping assay in the context of a clinical trial resulted in identification of pneumonia patients positive for the IATS-O11 serotype, and hence eligible for therapy with panobacumab (an investigational monoclonal antibody against the O-polysaccharide of serotype IATS-O11).

Pharmacokinetics and safety of panobacumab: specific adjunctive immunotherapy in critical patients with nosocomial Pseudomonas aeruginosa O11 pneumonia.

OBJECTIVES: Nosocomial Pseudomonas aeruginosa pneumonia remains a major concern in critically ill patients. We explored the potential impact of microorganism-targeted adjunctive immunotherapy in such patients. PATIENTS AND METHODS: This multicentre, open pilot Phase 2a clinical trial (NCT00851435) prospectively evaluated the safety, pharmacokinetics and potential efficacy of three doses of 1.2 mg/kg panobacumab, a fully human monoclonal anti-lipopolysaccharide IgM, given every 72 h in 18 patients developing nosocomial P. aeruginosa (serotype O11) pneumonia. RESULTS: Seventeen out of 18 patients were included in the pharmacokinetic analysis. In 13 patients receiving three doses, the maximal concentration after the third infusion was 33.9 ±â€Š8.0 µg/mL, total area under the serum concentration-time curve was 5397 ±â€Š1993 µg h/mL and elimination half-life was 102.3 ±â€Š47.8 h. Panobacumab was well tolerated, induced no immunogenicity and was detected in respiratory samples. In contrast to Acute Physiology and Chronic Health Evaluation II (APACHE II) prediction, all 13 patients receiving three doses survived, with a mean clinical resolution in 9.0 ±â€Š2.7 days. Two patients suffered a recurrence at days 17 and 20. CONCLUSIONS: These data suggest that panobacumab is safe, with a pharmacokinetic profile similar to that in healthy volunteers. It was associated with high clinical cure and survival rates in patients developing nosocomial P. aeruginosa O11 pneumonia. We concluded that these promising results warrant further trials.

An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties.

The antibody-mediated targeted delivery of interleukin-12 (IL12) to the EDB domain of fibronectin, a marker of angiogenesis, is a promising avenue for enhancing the therapeutic index of this anti-cancer cytokine. Previous experiments, based on sequential fusion of a single-chain IL12 derivative to the anti-EDB antibody fragment scFv(L19) had yielded a therapeutic fusion protein [IL12-scFv(L19)-FLAG], which displayed an impressive therapeutic activity in murine models of cancer, in spite of a tumor uptake, which was less efficient compared to the parental unmodified scFv(L19). In this article, we describe the comparative analysis of 3 recombinant fusion proteins comprising the scFv(L19) and IL12 moieties. One of them, in which the p40 and p35 form a covalent heterodimer and in which each subunit is fused to a molecule of scFv(L19), displays an excellent tumor targeting performance in vivo, as assessed by quantitative biodistribution analysis, and a potent anti-tumor effect, superior to the one of IL12-scFv(L19)-FLAG. These results may have a clinical impact, considering the fact that the tumor targeting ability of scFv(L19) in patients with cancer has been demonstrated using scintigraphic methods, and that 2 scFv(L19)-based antibody-cytokine fusion are currently entering clinical trials.

Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha.

The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of cytokines in cancer therapy consists of fusing them to monoclonal antibodies capable of a selective localization at the tumor site. We have constructed fusion proteins of interleukin-12 (IL-12) and tumor necrosis factor (TNF-alpha) with L19, an antibody fragment specific to the extradomain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. In this article, we have evaluated the tumor-targeting properties and the anticancer activities of combinations of the two antibody-cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-alpha. Although all fusion proteins were active in vitro, the triple fusion protein failed to localize to tumors in vivo and to show significant therapeutic effects. By contrast, the combination of IL-12-L19 and L19-TNF-alpha displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF-alpha (2 micro g + 2 micro g/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF-alpha appears to be synergistic in vivo.

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles.

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34. The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.