S100-alarmin-induced innate immune programming protects newborn infants from sepsis.

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however, the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic, epigenetic and immunological approaches, we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide, but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates, shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective, transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection.

A New Methodology for the Oxygen Measurement in Lung Tissue of an Aged Ferret Model Proves Hypoxia During COVID-19.

Oxygen as a key element has a high impact on cellular processes. Infection with a pathogen such as SARS-CoV-2 and following inflammation may lead to hypoxic conditions in tissue that impact cellular responses. To develop optimized translational in vitro models for a better understanding of physiologic and pathophysiologic oxygen conditions, it is a prerequisite to determine oxygen levels generated in vivo. Our study objective was the establishment of an invasive method for oxygen measurements using a luminescence-based microsensor to determine the dissolved oxygen in the lung tissue of ferrets as animal models for SARS-CoV-2 research. In analogy to humans, aged ferrets are more likely to show clinical signs after SARS-CoV-2 infection compared to young animals. To investigate oxygen levels during a respiratory viral infection, we intratracheally infected nine aged (3-year-old) ferrets with SARS-CoV-2. The aged SARS-CoV-2 infected ferrets showed mild to moderate clinical signs associated with prolonged viral RNA shedding until 14 days post infection (dpi). SARS-CoV-2 infected ferrets showed histopathologic lung lesion scores that significantly negatively correlated with oxygen levels in lung tissue. At 4 dpi, oxygen levels in lung tissue were significantly lower (mean %O2 of 3.89 ≙ ≈ 27.78 mmHg) compared to the negative control group (mean %O2 of 8.65 ≙ ≈ 61.4 mmHg). In summary, we succeeded in determining the pathophysiologic oxygen conditions in the lung tissue of aged SARS-CoV-2-infected ferrets. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). .

Alteration of cholesterol content and oxygen level in intestinal organoids after infection with Staphylococcus aureus.

The pathogenicity elicited by Staphylococcus (S.) aureus, one of the best-studied bacteria, in the intestine is not well understood. Recently, we demonstrated that S. aureus infection induces alterations in membrane composition that are associated with concomitant impairment of intestinal function. Here, we used two organoid models, induced pluripotent stem cell (iPSC)-derived intestinal organoids and colonic intestinal stem cell-derived intestinal organoids (colonoids), to examine how sterol metabolism and oxygen levels change in response to S. aureus infection. HPLC quantification showed differences in lipid homeostasis between infected and uninfected cells, characterized by a remarkable decrease in total cellular cholesterol. As the altered sterol metabolism is often due to oxidative stress response, we next examined intracellular and extracellular oxygen levels. Three different approaches to oxygen measurement were applied: (1) cell-penetrating nanoparticles to quantify intracellular oxygen content, (2) sensor plates to quantify extracellular oxygen content in the medium, and (3) a sensor foil system for oxygen distribution in organoid cultures. The data revealed significant intracellular and extracellular oxygen drop after infection in both intestinal organoid models as well as in Caco-2 cells, which even 48 h after elimination of extracellular bacteria, did not return to preinfection oxygen levels. In summary, we show alterations in sterol metabolism and intra- and extracellular hypoxia as a result of S. aureus infection. These results will help understand the cellular stress responses during sustained bacterial infections in the intestinal epithelium.

SARS-CoV-2 inactivation in laboratory animal tissues with 4% formaldehyde or 5% glutaraldehyde for transfer to biosafety level 1 laboratories.

The SARS-CoV-2 pandemic required the immediate need to transfer inactivated tissue from biosafety level (BSL)-3 to BSL-1 areas to enable downstream analytical methods. No validated SARS-CoV-2 inactivation protocols were available for either formaldehyde (FA)-fixed or glutaraldehyde (GA)-fixed tissues. Therefore, representative tissue from ferrets and hamsters was spiked with 2.2 × 106 tissue culture infectious dose 50% per ml (TCID50/ml) SARS-CoV-2 or were obtained from mice experimentally infected with SARS-CoV-2. SARS-CoV-2 inactivation was demonstrated with 4% FA or 5% GA at room temperature for 72 hours by a titer reduction of up to 103.8 TCID50/ml in different animal tissues with a maximum protein content of 100 µg/mg and a thickness of up to 10 mm for FA and 8 mm for GA. Our protocols can be easily adapted for validating the inactivation of other pathogens to allow for the transfer of biological samples from BSL-3 areas to BSL-1 laboratories.

Detrimental Role of Neutrophil Extracellular Traps during Dengue Virus Infection.

A recent article by Sung et al. identified the CLEC2 platelet receptor as an important factor of lethal dengue virus infection. Formation of neutrophil extracellular traps via crosstalk with CLEC5A and TLR2 neutrophils were ascribed a causative role in DENV infection. This provides new insights for the development of candidate broad-spectrum therapies against hemorrhagic virus infections.

Ferrets are valuable models for SARS-CoV-2 research.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in an ongoing pandemic with millions of deaths worldwide. Infection of humans can be asymptomatic or result in fever, fatigue, dry cough, dyspnea, and acute respiratory distress syndrome with multiorgan failure in severe cases. The pathogenesis of COVID-19 is not fully understood, and various models employing different species are currently applied. Ferrets can be infected with SARS-CoV-2 and efficiently transmit the virus to contact animals. In contrast to hamsters, ferrets usually show mild disease and viral replication restricted to the upper airways. Most reports have used the intranasal inoculation route, while the intratracheal infection model is not well characterized. Herein, we present clinical, virological, and pathological data from young ferrets intratracheally inoculated with SARS-CoV-2. Infected animals showed no significant clinical signs, and had transient infection with peak viral RNA loads at 4 days postinfection, mild to moderate rhinitis, and pulmonary endothelialitis/vasculitis. Viral antigen was exclusively found in the respiratory epithelium of the nasal cavity, indicating a particular tropism for cells in this location. Viral antigen was associated with epithelial damage and influx of inflammatory cells, including activated neutrophils releasing neutrophil extracellular traps. Scanning electron microscopy of the nasal respiratory mucosa revealed loss of cilia, shedding, and rupture of epithelial cells. The currently established ferret SARS-CoV-2 infection models are comparatively discussed with SARS-CoV-2 pathogenesis in mink, and the advantages and disadvantages of both species as research models for zoonotic betacoronaviruses are highlighted.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α.

Formation of neutrophil extracellular traps (NETs) is a two-faced innate host defense mechanism, which, on the one hand, can counteract microbial infections, but on the other hand, can contribute to massive detrimental effects on the host. Cholesterol depletion from the cellular membrane by Methyl-ß-cyclodextrin (MßCD) is known as one of the processes initiating NET formation. Since neutrophils mainly act in an inflammatory environment with decreased, so-called hypoxic, oxygen conditions, we aimed to study the effect of oxygen and the oxygen stress regulator hypoxia-inducible factor (HIF)-1α on cholesterol-dependent NET formation. Thus, murine bone marrow-derived neutrophils from wild-type and HIF-knockout mice or human neutrophils were stimulated with MßCD under normoxic (21% O2) compared to hypoxic (1% O2) conditions, and the formation of NETs were studied by immunofluorescence microscopy. We found significantly induced NET formation after treatment with MßCD in murine neutrophils derived from wild-type as well as HIF-1α KO mice at both hypoxic (1% O2) as well as normoxic (21% O2) conditions. Similar observations were made in freshly isolated human neutrophils after stimulation with MßCD or statins, which block the HMG-CoA reductase as the key enzyme in the cholesterol metabolism. HPLC was used to confirm the reduction of cholesterol in treated neutrophils. In summary, we were able to show that NET formation via MßCD or statin-treatment is oxygen and HIF-1α independent.

New Insights into Neutrophil Extracellular Trap (NETs) Formation from Porcine Neutrophils in Response to Bacterial Infections.

Actinobacillus pleuropneumoniae (A.pp, Gram negative) and Streptococcus (S.) suis (Gram positive) can cause severe diseases in pigs. During infection, neutrophils infiltrate to counteract these pathogens with phagocytosis and/or neutrophil extracellular traps (NETs). NETs consist of a DNA-backbone spiked with antimicrobial components. The NET formation mechanisms in porcine neutrophils as a response to both of the pathogens are not entirely clear. The aim of this study was to investigate whether A.pp (serotype 2, C3656/0271/11) and S. suis (serotype 2, strain 10) induce NETs by NADPH oxidase- or CD18-dependent mechanisms and to characterize phenotypes of NETs in porcine neutrophils. Therefore, we investigated NET induction in porcine neutrophils in the presence and absence of NET inhibitors and quantified NETs after 3 h. Furthermore, NETosis and phagocytosis were investigated by transmission electron microscopy after 30 min to characterize different phenotypes. A.pp and S. suis induce NETs that are mainly ROS-dependent. A.pp induces NETs that are partially CD18-dependent. Thirty minutes after infection, both of the pathogens induced a vesicular NET formation with only slight differences. Interestingly, some neutrophils showed only NET-marker positive phagolysosomes, but no NET-marker positive vesicles. Other neutrophils showed vesicular NETs and only NET-marker negative phagolysosomes. In conclusion, both of the pathogens induce ROS-dependent NETs. Vesicular NETosis and phagocytosis occur in parallel in porcine neutrophils in response to S. suis serotype 2 and A.pp serotype 2.

SARS-CoV-2-Specific Antibodies in Domestic Cats during First COVID-19 Wave, Europe.

We conducted a severe acute respiratory syndrome coronavirus 2 antibody seroprevalence study among >2,000 domestic cats from 4 countries during the first coronavirus disease wave in Europe. We found 4.4% seroprevalence using a virus neutralization test and 4.3% using a receptor-binding domain ELISA, demonstrating probable human-to-cat transmission.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

In vivo oxygen measurement in cerebrospinal fluid of pigs to determine physiologic and pathophysiologic oxygen values during CNS infections.

During infection and inflammation, a reduced oxygen level clearly affects cellular functions. Oxygen levels during CNS infections are unknown. Here we established and evaluated an in vivo measurement system to characterize the oxygen level in parallel with bacterial numbers (CFU/mL), the cell number and pH level inside the CSF of healthy compared to Streptococcus suis-infected pigs. The animals were anesthetized over a seven-hour period with isoflurane in air/oxygen at physiologic arterial partial pressure of oxygen. Oxygen levels in CSF of anesthetized pigs were compared to euthanized pigs. The detected partial pressure of oxygen in the CSF remained constant in a range of 47-63 mmHg, independent of the infection status (bacterial or cell number). In contrast, the pH value showed a slight drop during infection, which correlated with cell and bacterial number in CSF. We present physiologic oxygen and pH values in CSF during the onset of bacterial meningitis.

Scent dog identification of SARS-CoV-2 infections in different body fluids.

BACKGROUND: The main strategy to contain the current SARS-CoV-2 pandemic remains to implement a comprehensive testing, tracing and quarantining strategy until vaccination of the population is adequate. Scent dogs could support current testing strategies. METHODS: Ten dogs were trained for 8 days to detect SARS-CoV-2 infections in beta-propiolactone inactivated saliva samples. The subsequent cognitive transfer performance for the recognition of non-inactivated samples were tested on three different body fluids (saliva, urine, and sweat) in a randomised, double-blind controlled study. RESULTS: Dogs were tested on a total of 5242 randomised sample presentations. Dogs detected non-inactivated saliva samples with a diagnostic sensitivity of 84% (95% CI: 62.5-94.44%) and specificity of 95% (95% CI: 93.4-96%). In a subsequent experiment to compare the scent recognition between the three non-inactivated body fluids, diagnostic sensitivity and specificity were 95% (95% CI: 66.67-100%) and 98% (95% CI: 94.87-100%) for urine, 91% (95% CI: 71.43-100%) and 94% (95% CI: 90.91-97.78%) for sweat, 82% (95% CI: 64.29-95.24%), and 96% (95% CI: 94.95-98.9%) for saliva respectively. CONCLUSIONS: The scent cognitive transfer performance between inactivated and non-inactivated samples as well as between different sample materials indicates that global, specific SARS-CoV-2-associated volatile compounds are released across different body secretions, independently from the patient's symptoms. All tested body fluids appear to be similarly suited for reliable detection of SARS-CoV-2 infected individuals.

Ischaemic postconditioning reduces apoptosis in experimental jejunal ischaemia in horses.

BACKGROUND: Ischaemic postconditioning (IPoC) refers to brief periods of reocclusion of blood supply following an ischaemic event. This has been shown to ameliorate ischaemia reperfusion injury in different tissues, and it may represent a feasible therapeutic strategy for ischaemia reperfusion injury following strangulating small intestinal lesions in horses. The objective of this study was to assess the degree cell death, inflammation, oxidative stress, and heat shock response in an equine experimental jejunal ischaemia model with and without IPoC. METHODS: In this randomized, controlled, experimental in vivo study, 14 horses were evenly assigned to a control group and a group subjected to IPoC. Under general anaesthesia, segmental ischaemia with arterial and venous occlusion was induced in 1.5 m jejunum. Following ischaemia, the mesenteric vessels were repeatedly re-occluded in group IPoC only. Full thickness intestinal samples and blood samples were taken at the end of the pre-ischaemia period, after ischaemia, and after 120 min of reperfusion. Immunohistochemical staining or enzymatic assays were performed to determine the selected variables. RESULTS: The mucosal cleaved-caspase-3 and TUNEL cell counts were significantly increased after reperfusion in the control group only. The cleaved-caspase-3 cell count was significantly lower in group IPoC after reperfusion compared to the control group. After reperfusion, the tissue myeloperoxidase activity and the calprotectin positive cell counts in the mucosa were increased in both groups, and only group IPoC showed a significant increase in the serosa. Tissue malondialdehyde and superoxide dismutase as well as blood lactate levels showed significant progression during ischaemia or reperfusion. The nuclear immunoreactivity of Heat shock protein-70 increased significantly during reperfusion. None of these variables differed between the groups. The neuronal cell counts in the myenteric plexus ganglia were not affected by the ischaemia model. CONCLUSIONS: A reduced apoptotic cell count was found in the group subjected to IPoC. None of the other tested variables were significantly affected by IPoC. Therefore, the clinical relevance and possible protective mechanism of IPoC in equine intestinal ischaemia remains unclear. Further research on the mechanism of action and its effect in clinical cases of strangulating colic is needed.

Mechanism of drug extrusion by brain endothelial cells via lysosomal drug trapping and disposal by neutrophils.

The blood-brain barrier protects the brain against a variety of potentially toxic compounds. Barrier function results from tight junctions between brain capillary endothelial cells and high expression of active efflux transporters, including P-glycoprotein (Pgp), at the apical membrane of these cells. In addition to actively transporting drugs out of the cell, Pgp mediates lysosomal sequestration of chemotherapeutic drugs in cancer cells, thus contributing to drug resistance. Here, we describe that lysosomal sequestration of Pgp substrates, including doxorubicin, also occurs in human and porcine brain endothelial cells that form the blood-brain barrier. This is followed by shedding of drug-sequestering vesicular structures, which stay attached to the apical side of the plasma membrane and form aggregates ("barrier bodies") that ultimately undergo phagocytosis by neutrophils, thus constituting an as-yet-undescribed mechanism of drug disposal. These findings introduce a mechanism that might contribute to brain protection against potentially toxic xenobiotics, including therapeutically important chemotherapeutic drugs.

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

Survival of Streptococcus suis in Porcine Blood Is Limited by the Antibody- and Complement-Dependent Oxidative Burst Response of Granulocytes.

Bacteremia is a hallmark of invasive Streptococcus suis infections of pigs, often leading to septicemia, meningitis, or arthritis. An important defense mechanism of neutrophils is the generation of reactive oxygen species (ROS). In this study, we report high levels of ROS production by blood granulocytes after intravenous infection of a pig with high levels of S. suis-specific antibodies and comparatively low levels of bacteremia. This prompted us to investigate the working hypothesis that the immunoglobulin-mediated oxidative burst contributes to the killing of S. suis in porcine blood. Several S. suis strains representing serotypes 2, 7, and 9 proved to be highly susceptible to the oxidative burst intermediate hydrogen peroxide, already at concentrations of 0.001%. The induction of ROS in granulocytes in ex vivo-infected reconstituted blood showed an association with pathogen-specific antibody levels. Importantly, inhibition of ROS production by the NADPH oxidase inhibitor apocynin led to significantly increased bacterial survival in the presence of high specific antibody levels. The oxidative burst rate of granulocytes partially depended on complement activation, as shown by specific inhibition. Furthermore, treatment of IgG-depleted serum with a specific IgM protease or heat to inactivate complement resulted in >3-fold decreased oxidative burst activity and increased bacterial survival in reconstituted porcine blood in accordance with an IgM-complement-oxidative burst axis. In conclusion, this study highlights an important control mechanism of S. suis bacteremia in the natural host: the induction of ROS in blood granulocytes via specific immunoglobulins such as IgM.

Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Newborn infants have a high disposition to develop systemic inflammatory response syndromes (SIRSs) upon inflammatory or infectious challenges. Moreover, there is a considerable trafficking of hematopoietic cells to tissues already under noninflammatory conditions. These age-specific characteristics suggest a hitherto unappreciated crucial role of the vascular endothelium during the neonatal period. Here, we demonstrate that healthy neonates showed already strong endothelial baseline activation, which was mediated by a constitutively increased production of TNF-α. In mice, pharmacological inhibition of TNF-α directly after birth prevented subsequent fatal SIRS but completely abrogated the recruitment of leukocytes to sites of infection. Importantly, in healthy neonates, blocking TNF-α at birth disrupted the physiologic leukocyte trafficking, which resulted in persistently altered leukocyte profiles at barrier sites. Collectively, these data suggest that constitutive TNF-α-mediated sterile endothelial activation in newborn infants contributes to the increased risk of developing SIRS but is needed to ensure the postnatal recruitment of leukocytes to organs and interfaces.-Bickes, M. S., Pirr, S., Heinemann, A. S., Fehlhaber, B., Halle, S., Völlger, L., Willers, M., Richter, M., Böhne, C., Albrecht, M., Langer, M., Pfeifer, S., Jonigk, D., Vieten, G., Ure, B., von Kaisenberg, C., Förster, R., von Köckritz-Blickwede, M., Hansen, G., Viemann, D. Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.