2C5-surface modified nanocarriers targeting neutrophil extracellular traps

Background: Neutrophil extracellular traps (NETs) are composed of processed chromatin bound to granular and selected cytoplasmic proteins and released by neutrophils. NETs consist of smooth filaments composed of stacked nucleosomes. Fully hydrated NETs have a cloud-like appearance and occupy a space 10-15-fold larger than the volume of the cells they originate from. DNases are the enzymes that cleave extracellular DNA including NETs. Together with their protective role in microbial infections, NETs are involved in multiple pathological processes and represent key events in a variety of pathologies including cancer, autoimmunity, and cardiovascular disease. Sites of NETs concentration are dangerous for the host if the process of NETs formation becomes chronic or the mechanism of NETs removal does not work. NETosis has been linked to the development of periodontitis, cystic fibrosis, type 2 diabetes, COVID-19 or rheumatoid arthritis as well as cancer progression. Purpose(s): Thus, the destruction of NETs is of primary significance in many pathologies. In our approach, we are focusing on mimicking one of the natural mechanisms of destroying excessive NETs by delivering deoxyribonuclease I to the specific site of pathological NETs accumulation by modifying the nanoparticles using an anti-nucleosome monoclonal antibody (2C5). The antibody is specific to nucleosomes and can recognize histones in NETs. DNase I is U.S. Food and Drug Administration (FDA)-approved active component and is commonly used in therapeutic methods of modern medicine for cystic fibrosis to clear extracellular DNA fibers in the lungs and systemic lupus erythematosus. Recent findings have also shown the effectiveness of DNase I in the digestion of NETs. However, the low serum stability and fast deactivation by environmental stimuli have been considered as the limiting factors for clinical applications of DNase I, which can be overcome by its targeted specific delivery in pharmaceutical nanocarriers. Method(s): In this study, we generate NETs in vitro using human neutrophils and HL-60 cells differentiated into granulocyte-like cells. We used interleukin-8, lipopolysaccharide from E.Coli (LPS), phorbol myristate acetate (PMA), and calcium ionophore A23187 (CI) to generate the NETs. We confirmed the specificity of 2C5 toward NETs by ELISA, which showed that it binds to NETs with the specificity like that for purified nucleohistone substrate. We further utilized that feature to create two delivery systems (liposomes and micelles) for DNAse I enzyme to destroy NETs, which was confirmed by staining NETs with SYTOX Green dye and followed by flow cytometric measurements and microscopic images. Conclusion(s): Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies. Also, we proved that our carrier can successfully deliver DNase to NETs to provide their degradation.

COVID-19 neutrophils show elevated migration and NETosis, which is reduced by PI3K inhibition

Rational: Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics. Cell functions are interwoven pathways, so understanding the effect of COVID-19 across neutrophil function may identify therapeutic targets. We examined neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia (CAP) patients. Method(s): Isolated neutrophils underwent ex vivo analyses for migration, phagocytosis and NETosis, and the effect of PI3K inhibition. Circulating DNAse 1 activity and levels of cfDNA were measured. Result(s): Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397, A) and NETosis (p=0.0366, B), but impaired phagocytosis (p=0.0236, C) associated with impaired ROS generation (p<0.0001). COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001, D). Ex vivo inhibition of PI3K gamma and delta reduced NET release by COVID-19 neutrophils (p=0.0156). Conclusion(s): COVID-19 is associated with neutrophil dysfunction across all main effector functions, with elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function via PI3k may help modulate COVID-19 severity. (Figure Presented).

USE OF ALTEPLASE AND DORNASE FOR LYSIS OF COVID-19-RELATED LOCULATED PLEURAL EFFUSION

SESSION TITLE: Management of COVID-19-Induced Complications SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/19/2022 12:45 pm - 1:45 pm INTRODUCTION: We discuss a case of successful use of alteplase and dornase per MIST II protocol for the management of a loculated pleural effusion secondary to COVID-19 pneumonia. CASE PRESENTATION: 52 year old male was initially admitted for MRSA bacteremia and began appropriate antibiotic therapy. His chest radiograph on presentation was unremarkable. Seven days into his hospital course he tested positive for COVID-19 pneumonia, and developed increasing shortness of breath and escalating oxygen requirements. At this time he had a large loculated left sided pleural effusion on chest computed tomography. A pigtail catheter was placed with removal of 600ml of cloudy yellowish fluid. Follow-up CXR showed slight interval improvement, however a large loculated effusion remained. Pleural fluid studies was exudative, lymphocytic predominant (78%) with elevated pleural fluid lactate dehydrogenase of 786 U/L, pH 8.0, and glucose 97mg/dl. Additional pleural fluid workup was unremarkable, including negative cultures, AFB staining, and benign cytology. After other known causes of lymphocyte predominant pleural effusion were ruled out, and following review of the current medical literature, the conclusion was made that his effusion was most likely related to COVID-19. The decision was made to attempt lysis of the loculations with alteplase and dornase per MIST II protocol. This resulted in significant chest tube output (totaling 3480ml additional output over the ensuing days) as well as marked improvement in chest imaging. The protocol was continued for 3 days which the patient tolerated well overall. DISCUSSION: COVID-19 related pleural effusions occur with an incidence of about 7.3% of cases with an overall lag time of 11 days from symptom onset. Based on observational studies, these pleural effusions are unilateral in 66.8% of cases with a lymphocyte or neutrophilic predominance and significantly elevated pleural fluid to serum LDH ratio. The differential for exudative lymphocyte predominant pleural effusions with elevated LDH include malignancy, rheumatoid effusion, tuberculosis, and viral infections. The pleural studies workup was unremarkable for these conditions. The MIST-2 protocol was followed per the original study, with instillation of tPA 10mg via pigtail catheter which was clamped for 1 hour, opened to drain for 1 hour, then repeated with dornase 5mg. To the best of our knowledge, this is the first documented case of using MIST 2 protocol for a loculated pleural effusion related to COVID-19. CONCLUSIONS: COVID-19 related loculated pleural effusion is an infrequent occurrence that present as lymphocyte predominant exudative that can loculate with elevated lactate dehydrogenase. This is the first case of using alteplase and dornase for its management and we have demonstrated that it can be both a safe and effective method. Additional prospective studies are needed to further investigate this method. Reference #1: Chong WH, Saha BK, Conuel E, Chopra A. The incidence of pleural effusion in COVID-19 pneumonia: State-of-the-art review. Heart Lung. 2021;50(4):481-490. doi:10.1016/j.hrtlng.2021.02.015 Reference #2: Ahmadinejad Z, Salahshour F, Dadras O, Rezaei H, SeyedAlinaghi S. Pleural Effusion as a Sign of Coronavirus Disease 2019 (COVID-19) Pneumonia: A Case Report. Infect Disord Drug Targets. 2021;21(3):468-472. doi: 10.2174/1871526520666200609125045. PMID: 32516107. Reference #3: Rahman, N, et al. Intrapleural Use of Tissue Plasminogen Activator and DNase in Pleural Infection. N Engl J Med 2011;365:518-526. DOI: 10.1056/NEJMoa1012740 DISCLOSURES: No relevant relationships by Zachary Chandler No relevant relationships by James Cury No relevant relationships by Peter Staiano No relevant relationships by Daniel Weigle

ENTEROCOCCUS EMPYEMA FOLLOWING EXTRACORPEAL MEMBRANE OXYGENATION (ECMO) FOR COVID-19 ARDS: A CASE REPORT

SESSION TITLE: Management of COVID-19-Induced Complications SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/19/2022 12:45 pm - 1:45 pm INTRODUCTION: Multiple pleural complications have been well described during COVID-19 infection including pneumothorax, pleural effusion and empyema. While many infections have been described as complications, here we present a case of empyema secondary to Enterococcus faecium in a patient with COVID-19 after Extra-corporeal membrane oxygenation (ECMO). CASE PRESENTATION: A 33-year-old male presented with acute respiratory distress syndrome secondary to COVID-19. He was intubated 10 days after symptom onset and subsequently placed on veno-venous ECMO, with reconfiguration to add an arterial return limb and eventually pulmonary artery return limb. During his care he was treated with remdesivir, dexamethasone and tocilizumab. His course was complicated by right heart failure requiring mechanical support, acute renal failure requiring hemodialysis, superior vena cava thrombus, multi-drug resistant Pseudomonas, Enterococcus faecalis, Klebsiella and methicillin sensitive Staphylococcus aureus infections. Eventually he was removed from ECMO on day 130. The patient remained in the ICU on positive pressure ventilation via tracheostomy. He eventually developed worsening respiratory status as well as signs concerning for an emerging infection. Broad spectrum antibiotics were initiated, and a CT chest/abdomen/pelvis was obtained that showed right pleural effusion with concern for empyema. Pleural sampling was consistent with empyema with glucose <5 mg/dL, pH <7.2, lactate dehydrogenase >200 U/L and an elevated neutrophil count. A percutaneously placed 14 french chest tube was placed and pleural irrigation with normal saline was trialed given patient need for continuous systemic anticoagulation. However, this did not sufficiently resolve the empyema and patient was started on pleural TPA/Dornase with close monitoring while on anticoagulation with clinicoradiographic improvement after a total of 6 days of therapy. Cultures eventually speciated as Enterococcus faecium and he was continued on a 6 week course of ampicillin for his empyema. DISCUSSION: Classically, Enterococcus empyema has been primarily linked with intra-abdominal infections which was not found in our patient and has not been correlated with COVID-19 infection or ECMO. Additionally, there is a significant paucity of data with regards to safety of TPA/Dornase pleural irrigation use while patients are on full dose systemic anticoagulation as was our patient. In this case he required two 3-day courses of TPA/Dornase which was tolerated well without significant complication. CONCLUSIONS: Here we describe a rare causative organism of empyema that has not been previously described in the literature as associated with COVID-19 or ECMO. Additionally, we demonstrate the safety of intra-pleural TPA/Dornase in this patient on full dose anticoagulation which is a frequent consideration when determining the method of treating empyema in complex medical patients. Reference #1: Ayad S, Gergis K, Elkattawy S, et al. Loculated empyema and SARS-COV-2 infection: A report of two cases and review of the literature. European journal of case reports in internal medicine. July 2021;8(7):002706. doi:10.12890/2021_002706. Reference #2: Bergman R, Tjan DH, Schouten MA, Haas LE, van Zanten AR. Pleural Enterococcus faecalis empyema: an unusual case. Infection. Feb 2009;37(1):56-9. doi:10.1007/s15010-007-6359-6 Reference #3: Rahman NM, Al. E, Author Affiliations From the United Kingdom Clinical Research Collaboration Oxford Respiratory Trials Unit and Oxford Pleural Diseases Unit, et al. Intrapleural use of tissue plasminogen activator and DNase in pleural infection: Nejm. New England Journal of Medicine. Nov 2011;365:518-526. doi:10.1056NEJMoa1012740. DISCLOSURES: No relevant relationships by Joshua Boster No relevant relationships by Mary Gadarowski No relevant relationships by Stephen Goertzen No relevant relationships by Amanda Hall No relevant relat onships by Erik Manninen

Isolation, biochemical characterization and safety screening of potential probiotic lactic acid bacteria from spontaneously fermented cereal products from Western Kenya

The global demand for non-dairy beverages has sky rocketed especially so during this Covid-19 pandemic for potential health benefits. Development of probiotic strains from fermented cereal and legumes with the ability to grow well and adapt to gastrointestinal conditions at the same time possess high therapeutic ability will be a great achievement. This study aimed at isolating and screening probiotic potential Lactic Acid Bacteria (LAB) involved in traditional fermentation of cereals (maize, sorghum and millet). A total of ten isolates were obtained from the cereals out of which five isolates that met preliminary attributes of probiotic bacteria were selected for further investigation. Two isolates SPU2 and FPU1 were found to survive a low pH which is a desirable attribute for the survival of probiotic bacteria in the gut. MPU1, FPU1 and SPU2 are possible thermophiles and can survive at low pH and moderate high salt concentration. The enzymes DNase and gelatinase used to test pathogenicity of a microorganism were not produced by all the isolates in this study. The isolates recorded a high susceptibility to the eight antibiotics. This study also revealed that the tested isolates have the ability to grow well even at the minimum tested pH of 1.0 for 1 and 2 h of incubation, respectively. Most isolates were resistant to 0.3% bile concentration with over 92% survival. FPU1 was more resistant at bile concentration of 1% than all the rest while MPU1 was most resistant at 2% bile salt. Traditionally fermented cereals are potential sources of safe bacteria that can be tried in the production of functional foods.