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Background Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α‐1,3 Gal (Galactose‐alpha‐1,3‐galactose) causing hyperacute rejection, also Neu5Gc (N‐glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross‐reacted with swine leucocyte antigen class I (SLA‐I). We previously demonstrated efficient generation of pigs with multiple xeno‐transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi‐transgenic background further reduces antibody deposition and complement activation. Methods Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one‐ to four‐fold knockout animals, and from multi‐transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four‐fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. Results Pigs were generated carrying four‐fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi‐transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. Conclusion We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Background Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48 hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non‐ischemic perfusion for our ongoing pig‐to‐baboon cardiac xenotransplantation project. Methods Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1‐ KO/hCD46/hTBM) as donors and captive‐bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n = 5) and cold non‐ischemic continuous perfusion (CP; n = 9) with an oxygenated albumin‐containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti‐non‐Gal‐antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient’s kidney, liver and coagulation functions. Results In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti‐non‐Gal‐antibodies were similar in recipients receiving grafts from either IC or CP preservation. Conclusions While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi‐organ failure in more than half of the xenotransplantation experiments. In contrast, cold non‐ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long‐term results after cardiac xenotransplantation.

The human skin microbiome has recently become a focus for both the dermatological and cosmetic fields. Understanding the skin microbiota, that is the collection of vital microorganisms living on our skin, and how to maintain its delicate balance is an essential step to gain insight into the mechanisms responsible for healthy skin and its appearance. Imbalances in the skin microbiota composition (dysbiosis) are associated with several skin conditions, either pathological such as eczema, acne, allergies or dandruff or non‐pathological such as sensitive skin, irritated skin or dry skin. Therefore, the development of approaches which preserve or restore the natural, individual balance of the microbiota represents a novel target not only for dermatologists but also for skincare applications. This review gives an overview on the current knowledge on the skin microbiome, the currently available sampling and analysis techniques as well as a description of current approaches undertaken in the skincare segment to help restoring and balancing the structure and functionality of the skin microbiota.

In the context of xenotransplantation, in ischemia/reperfusion injury as well as in cardiovascular research, the study of the fascinating interplay between endothelial cells (EC) and the plasma cascade systems often requires in vitro models. Blood vessels are hardly reproducible with standard flat-bed culture systems and flow-plate assays are limited in their low surface-to-volume ratio which impedes the study of the anticoagulant properties of the endothelial cells. According to the 3R regulations (reduce, replace and refine animal experimentation) we developed a closed circuit microfluidic in vitro system in which endothelial cells are cultured in 3D round section microchannels and subjected to physiological, pulsatile flow. In this study, a 3D monolayer of porcine aortic EC was perfused with human serum to mimic a xenotransplantation setting. Complement as well as EC activation was assessed in the presence or absence of complement inhibitors showing the versatility of the model for drug testing. Complement activation products as well as E-selectin expression were detected and visualized in situ by high resolution confocal microscopy. Furthermore, porcine pro-inflammatory cytokines as well as soluble complement components in the recirculating fluid phase were detected after human serum perfusion providing a better overview of the artificial vascular environment.

GTKO, hCD46, and HLA-E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human-anti-pig rejection reactions and improve myocardial function after xenotransplantation.

Skin cleansing represents a process of mechanical and chemical removal of dirt, pollutants as well as microbiota from the skin. While skin cleansing can help maintain good health, protect us from infections, illnesses and ailments, skin cleansing can also strip away lipids and moisture from the skin, leading to irritation, barrier impairment and disturbance of the delicate cutaneous microbiome. This study investigated how skin cleansing impacts skin’s microbial composition. Thirty Caucasian women were enrolled in a placebo controlled clinical study where participants applied on their volar forearms a liquid body wash twice daily for 1 week in order to mimic frequent showering. Skin microbiome samples were collected by swabbing at defined timepoints and 16S rRNA sequencing was performed. Using “reference frames”, we could identify shifts in the microbial composition and several microbiota were identified as being characteristically associated with the presence of saccharide isomerate, a well-known skin moisturizer. The microbial shift was quite immediate, and we could observe it already at 1 h post cleansing. Interestingly, the new microbial composition reached a certain dynamic equilibrium at day 1 which was then maintained until the end of the study. Paracoccus marcusii, a potentially beneficial carotenoid-producer microorganism, was enriched by the active treatment and, at the same time, the abundance of several potential pathogenic taxa, Brevibacterium casei and Rothia mucilaginosa, diminished.