Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. Methodology/Principal Findings: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. Conclusions: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.National Institutes of Health (NIH)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)NeoPharm, In

Effect of Carbon Dioxide Pneumo-peritoneum in Coagulation Profile of Patients undergoing Laparoscopic Cholecystectomy: A Prospective Cohort Study

Introduction: Laparoscopic Cholecystectomy (LC) is done under general anaesthesia with the patient in a reverse Trendelenburg position and with pressurised carbon dioxide in the peritoneum. This can induce venous stasis in the lower extremities and may affect the balance in the coagulation and fibrinolysis system, thereby thrombo-embolic complications. Aim: To investigate the effects of carbon dioxide pneumo-peritoneum on the coagulation system of patients undergoing LC. Materials and Methods: A prospective longitudinal study was carried out from January 2021 to June 2021 among patients aged 18 to 60 years who attended the Surgery Department at Regional Institute of Medical Sciences, Imphal, Manipur, India and were diagnosed with gallstone disease and subsequently underwent LC. Independent variables like age, sex, religion, pre-operative prothrombin time, platelet count, activated Partial Thromboplastin Time (aPTT), and International Normalised Ratio (INR). Outcome variables comprised complications, post-operative prothrombin time, platelet count, aPTT, and INR. Data collected were analysed using Statistical Package for Social Sciences(SPSS) version 21.0. Paired t-tests were employed to test the association between mean values of post-operative and pre-operative PT, aPTT, INR, etc. A p-value of less than 0.05 was considered statistically significant. Results: The study enrolled 71 patients who encountered LC with carbon dioxide pneumo-peritoneum, including 18 male and 54 female patients. Maximum number of patients (28, 38.9%) fell into the 41 to 50 years age group. There was no significant difference in the mean value of prothrombin time (p=0.150) and INR (p=0.437) measured between the pre-operative and post-operative periods. Conclusion: LC is a safe procedure without clinically significant changes in the coagulation profile

Effect of systemic IL13-PE sensitization on the development of pulmonary fibrosis and the subsequent intranasal IL13-PE therapy.

<p>Mice were sensitized with IL13-PE via an intraperitoneal immunization protocol. Controls received saline alone. One week after last dose of IL13-PE or saline, all mice were injected i.t. with bleomycin. Twenty-one days after the induction of pulmonary fibrosis, treatment started with saline or IL13-PE intranasal instillation. At day 28 after bleomycin, lungs were collected for histopathological or biochemical assessment of pulmonary fibrosis. Magnification 100× (<b>A</b>). Hydroxyproline content in lung homogenate from saline (closed bars) or IL13-PE (hatched bars) sensitized animals are shown (<b>B</b>).</p

IL13-PE cytotoxicity toward an IL-13Rα2-expressing tumor cell line in the presence of mouse sera from various groups of treated mice^a.

a<p>Mice were systemically sensitized to IL13-PE as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008721#s4" target="_blank">Materials and Methods</a> section. Prior to bleomycin challenge, blood samples were removed from the control (saline) and IL13-PE-treated groups (ie. the pre-bleomyin sample). Mice received bleomycin and 21 days later they were randomized to treatment groups, which received one of saline alone, 200 ng/dose of IL13-PE, 500 ng/dose of IL13-PE, or 1000 ng/dose of IL13-PE. Sera was removed from each group at day 28 after 4 treatments of saline or IL13-PE. For this cytotoxicity assay, 1×10⁴ tumor cells were cultured with IL13-PE and serum sample for 20 hr at 37°C, pulsed with 1 µCi of [³H]-leucine and further incubated for 4 hr. Cells were harvested and counted as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008721#s4" target="_blank">Materials and Methods</a> section.</p>b<p>IC₅₀ is the concentration of IL13-PE at which a 50% inhibition of protein synthesis occurs in IL13-PE treated tumor cells compared with untreated tumor cells.</p

<i>P. aeruginosa</i> infection induced an antibody response against PE and IL13-PE.

<p><b>A</b>: IgA levels in BAL; <b>B</b>: IgG2a in BAL; <b>C</b>: IgA in serum. C57Bl/6 mice were infected by oropharingeal route with one of a range of bacterial doses and their bronchoalveolar fluid (BAL) and blood were collected at 14 days post infection. Antibody levels were evaluated by ELISA using plates coated with PE (open bars) or IL13-PE (closed bars). Statistical comparisons were determined between infected and non-infected groups with the same coating on the ELISA plate. *p≤0.05 compared with biologic sample added to PE-coated wells. ^#p≤0.05 compared with biologic sample added to IL13-PE-coated wells.</p