These results provide evidence for the activity of phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.

Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F OEGMA) but a comparable degree of polymerization (DP ∼ 120). Increase of both F OEGMA and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.

Background and Purpose: Cell-free DNA (cfDNA) and endogenous deoxyribonuclease activity are opposing mediators and might influence the inflammatory response following acute ischemic stroke. In this cohort study, we investigated the relation between these markers, circulating inflammatory mediators and clinical course including occurrence of stroke-associated infections (SAI) in patients with acute stroke. Methods: Ninety-two patients with stroke due to large vessel occlusion undergoing mechanical thrombectomy were prospectively recruited at Hannover Medical School from March 2018 to August 2019. Deoxyribonuclease activity, cfDNA, damage-associated molecular patterns, and circulating cytokines were measured in venous blood collected immediately before mechanical thrombectomy and 7 days later. Reperfusion status was categorized (sufficient/insufficient). Clinical outcome was evaluated using the modified Rankin Scale after 90 days, where a score of 3 to 6 was considered unfavorable. To validate findings regarding SAI, another stroke cohort (n=92) was considered with blood taken within 24 hours after stroke onset. Results: Patients with unfavorable clinical outcome had higher cfDNA concentrations. After adjustment for confounders (Essen Stroke Risk Score, National Institutes of Health Stroke Scale, and sex), 7-day cfDNA was independently associated with clinical outcome and especially mortality (adjusted odds ratio: 3.485 [95% CI, 1.001–12.134] and adjusted odds ratio: 9.585 [95% CI, 2.006–45.790]). No association was found between reperfusion status and cfDNA or deoxyribonuclease activity. While cfDNA concentrations correlated positively, deoxyribonuclease activity inversely correlated with distinct biomarkers. Baseline deoxyribonuclease activity was lower in patients who developed SAI compared with patients without SAI. This association was confirmed after adjustment for confounding factors (adjusted odds ratio: 0.447 [95% CI, 0.237–0.844]). In cohort 2, differences of deoxyribonuclease activity between patients with and without SAI tended to be higher with higher stroke severity. Conclusions: The interplay of endogenous deoxyribonuclease activity and cfDNA in acute stroke entails interesting novel diagnostic and potential therapeutic approaches. We confirm an independent association of cfDNA with a detrimental clinical course after stroke due to large vessel occlusion. This study provides first evidence for lower endogenous deoxyribonuclease activity as risk factor for SAI after severe stroke.

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond.

Swine stocks are endemically infected with the major porcine pathogen Streptococcus (S.) suis. The factors governing the transition from colonizing S. suis residing in the tonsils and the exacerbation of disease have not yet been elucidated. We analyzed the sudden death of fattening pigs kept under extensive husbandry conditions in a zoo. The animals died suddenly of septic shock and showed disseminated intravascular coagulopathy. Genotypic and phenotypic characterizations of the isolated S. suis strains, a tonsillar isolate and an invasive cps type 2 strain, were conducted. Isolated S. suis from dead pigs belonged to cps type 2 strain ST28, whereas one tonsillar S. suis isolate harvested from a healthy animal belonged to ST1173. Neither S. suis growth, induction of neutrophil extracellular traps, nor survival in blood could explain the sudden deaths. Reconstituted blood assays with serum samples from pigs of different age groups from the zoo stock suggested varying protection of individuals against pathogenic cps type 2 strains especially in younger pigs. These findings highlight the benefit of further characterization of the causative strains in each case by sequence typing before autologous vaccine candidate selection.