Tackling the Root Cause of Surface-Induced Coagulation: Inhibition of FXII Activation to Mitigate Coagulation Propagation and Prevent Clotting.

Factor XII (FXII) is a zymogen present in blood that tends to adsorb onto the surfaces of blood-contacting medical devices. Once adsorbed, it becomes activated, initiating a cascade of enzymatic reactions that lead to surface-induced coagulation. This process is characterized by multiple redundancies, making it extremely challenging to prevent clot formation and preserve the properties of the surface. In this study, a novel modulatory coating system based on C1-esterase inhibitor (C1INH) functionalized polymer brushes, which effectively regulates the activation of FXII is proposed. Using surface plasmon resonance it is demonstrated that this coating system effectively repels blood plasma proteins, including FXII, while exhibiting high activity against activated FXII and plasma kallikrein under physiological conditions. This unique property enables the modulation of FXII activation without interfering with the overall hemostasis process. Furthermore, through dynamic Chandler loop studies, it is shown that this coating significantly improves the hemocompatibility of polymeric surfaces commonly used in medical devices. By addressing the root cause of contact activation, the synergistic interplay between the antifouling polymer brushes and the modulatory C1INH is expected to lay the foundation to enhance the hemocompatibility of medical device surfaces.

Profiling of time-dependent human plasma protein adsorption on non-coated and heparin-coated oxygenator membranes.

Patients with severe lung diseases are highly dependent on lung support systems. Despite many improvements, long-term use is not possible, mainly because of the strong body defence reactions (e.g. coagulation, complement system, inflammation and cell activation). The systematic characterization of adsorbed proteins on the gas exchange membrane of the lung system over time can provide insights into the course of various defence reactions and identify possible targets for surface modifications. Using comprehensive mass spectrometry analyses of desorbed proteins, we were able to identify for the first time binding profiles of over 500 proteins over a period of six hours on non-coated and heparin-coated PMP hollow fiber membranes. We observed a higher degree of remodeling of the protein layer on the non-coated membrane than on the coated membrane. In general, there was a higher protein binding on the coated membrane with exception of proteins with a heparin-binding site. Focusing on the most important pathways showed that almost all coagulation factors bound in higher amounts to the non-coated membranes. Furthermore, we could show that the initiator proteins of the complement system bound stronger to the heparinized membranes, but the subsequently activated proteins bound stronger to the non-coated membranes, thus complement activation on heparinized surfaces is mainly due to the alternative complement pathway. Our results provide a comprehensive insight into plasma protein adsorption on oxygenator membranes over time and point to new ways to better understand the processes on the membranes and to develop new specific surface modifications.

Delivery of synthetic mRNAs for tissue regeneration.

In recent years, nucleic acid-based therapeutics have gained increasing importance as novel treatment options for disease prevention and treatment. Synthetic messenger RNAs (mRNAs) are promising nucleic acid-based drugs to transiently express desired proteins that are missing or defective. Recently, synthetic mRNA-based vaccines encoding viral proteins have been approved for emergency use against COVID-19. Various types of vehicles, such as lipid nanoparticles (LNPs) and liposomes, are being investigated to enable the efficient uptake of mRNA molecules into desired cells. In addition, the introduction of novel chemical modifications into mRNAs increased the stability, enabled the modulation of nucleic acid-based drugs, and increased the efficiency of mRNA-based therapeutic approaches. In this review, novel and innovative strategies for the delivery of synthetic mRNA-based therapeutics for tissue regeneration are discussed. Moreover, with this review, we aim to highlight the versatility of synthetic mRNA molecules for various applications in the field of regenerative medicine and also discuss translational challenges and required improvements for mRNA-based drugs.

The novel Candida albicans transporter Dur31 Is a multi-stage pathogenicity factor.

Candida albicans is the most frequent cause of oral fungal infections. However, the exact pathogenicity mechanisms that this fungus employs are largely unknown and many of the genes expressed during oral infection are uncharacterized. In this study we sought to functionally characterize 12 previously unknown function genes associated with oral candidiasis. We generated homozygous knockout mutants for all 12 genes and analyzed their interaction with human oral epithelium in vitro. Eleven mutants caused significantly less epithelial damage and, of these, deletion of orf19.6656 (DUR31) elicited the strongest reduction in pathogenicity. Interestingly, DUR31 was not only involved in oral epithelial damage, but in multiple stages of candidiasis, including surviving attack by human neutrophils, endothelial damage and virulence in vivo. In silico analysis indicated that DUR31 encodes a sodium/substrate symporter with 13 transmembrane domains and no human homologue. We provide evidence that Dur31 transports histatin 5. This is one of the very first examples of microbial driven import of this highly cytotoxic antimicrobial peptide. Also, in contrast to wild type C. albicans, dur31Δ/Δ was unable to actively increase local environmental pH, suggesting that Dur31 lies in the extracellular alkalinization hyphal auto-induction pathway; and, indeed, DUR31 was required for morphogenesis. In agreement with this observation, dur31Δ/Δ was unable to assimilate the polyamine spermidine.

Embryonated chicken eggs as alternative infection model for pathogenic fungi.

Embryonated eggs have been used as infection models for decades in virology and bacteriology. However, they can also be used as an attractive alternative infection model for studying fungal pathogenesis. Here, we discuss some general aspects which need to be considered when working with embryonated eggs as infection models. Furthermore, we provide detailed protocols and technical tips for infection of embryonated eggs with Aspergillus fumigatus and Candida albicans via the chorioallantois membrane, as well as sampling methods for downstream analyses.

Pathogenesis of Candida albicans infections in the alternative chorio-allantoic membrane chicken embryo model resembles systemic murine infections.

Alternative models of microbial infections are increasingly used to screen virulence determinants of pathogens. In this study, we investigated the pathogenesis of Candida albicans and C. glabrata infections in chicken embryos infected via the chorio-allantoic membrane (CAM) and analyzed the virulence of deletion mutants. The developing immune system of the host significantly influenced susceptibility: With increasing age, embryos became more resistant and mounted a more balanced immune response, characterized by lower induction of proinflammatory cytokines and increased transcription of regulatory cytokines, suggesting that immunopathology contributes to pathogenesis. While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos. IL-22 and IL-17A were only upregulated after the peak mortality in the chicken embryo model occurred; thus, the role of the Th17 response in this model remains unclear. Abscess formation occurs frequently in murine models, whereas the avian response was dominated by granuloma formation. Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines. However, fungal burden did not correlate with virulence and for few mutants like bcr1Δ and tec1Δ different outcomes in survival compared to murine infections were observed. C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections. These data suggest that the pathogenesis of C. albicans infections in the chicken embryo model resembles systemic murine infections but also differs in some aspects. Despite its limitations, it presents a useful alternative tool to pre-screen C. albicans strains to select strains for subsequent testing in murine models.

Embryonated eggs as an alternative infection model to investigate Aspergillus fumigatus virulence.

Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the "gold standard" for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity.

Identifying infection-associated genes of Candida albicans in the postgenomic era.

The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.

Are heart valves from donors over 65 years of age morphologically suitable for transplantation?

Since there is no upper age limit for general organ donation, unlike heart valve donation, and since a quarter of all organ donors are 65 years and older, we examined whether the heart valves from these donors are suitable as allografts. In the period 1999-2004 the aortic valve and pulmonary valve of 100 organ donors above 65 years of age were examined to establish whether they would have been suitable as valve grafts. To compare the valve grafts above and below the age limit of 65 years, we used data on the aortic and pulmonary valves of 380 organ donors below the age limit in the same time period. Examination of the 200 heart valves showed that - just like valves from donors below the age limit - 100 of them would have met the medical quality standards for transplantation, which discriminate among optimal, suitable and unsuitable tissue morphology. The morphological suitability of the aortic valves decreases rapidly during the 4th decade of life and near to the age limit only 6% of them are accepted as grafts. The rate of potentially acceptable aortic valve grafts from organ donors aged over 65 years of 15% is also small. By contrast, the pulmonary valves are not affected by age-related tissue changes that might reduce their transplantability. The predominant majority (85%) of potential pulmonary valve grafts from organ donors over 65 years of age fulfilled the acceptance criteria, half of them (48%) even showing good tissue quality. In light of these results the age limit was raised to 70 years in 2005.

How large is the organ donor potential in Germany? Results of an analysis of data collected on deceased with primary and secondary brain damage in intensive care unit from 2002 to 2005.

The number of potential organ donors depends on various factors, among which the number of deceased with primary or secondary brain damage is the most decisive. In the north-east donor region of Germany with 7.69 million inhabitants, 2019 cases of deceased with primary or secondary brain damage were reported by 136 intensive care units during 2002-2005. In a study, 64% of these deceased were identified as potential donors. This represents 40.7 potential donors per million inhabitants. It can be concluded that in the other donor regions of Germany a comparable number of potential donors exists, yet not all possible donors are being detected and referred. The conversion rate (percentage of potential donors who become effective donors) in the years 2002-2005 was 47%. The main reason for the conversion rate being so low was the large number of relatives who declined an organ donation (73%). More than 90% of the relatives in the north-east region did not know the deceased's will in the acute situation. From our point of view the high refusal rate can be decreased mainly by two measures: improvement of the family approach and integrating the topic of organ donation into schools' curricula.