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Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans , a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans , candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans , usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer. Candida albicans , usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.

Diffusion and mobility are essential for cellular functions, as molecules are usually distributed throughout the cell and have to meet to interact and perform their function. This also involves the cytosolic migration of cellular organelles. However, observing such diffusion and interaction dynamics is challenging due to the high spatial and temporal resolution required and the accurate analysis of the diffusional tracks. The latter is especially important when identifying anomalous diffusion events, such as directed motions, which are often rare. Here, we investigate the migration modes of peroxisome organelles in the cytosol of living cells. Peroxisomes predominantly migrate randomly, but occasionally they bind to the cell's microtubular network and perform directed migration, which is difficult to quantify, and so far, accurate analysis of switching between these migration modes is missing. We set out to solve this limitation by experiments and analysis with high statistical accuracy. Specifically, we collect temporal diffusion tracks of thousands of individual peroxisomes in the HEK 293 cell line using two-dimensional spinning disc fluorescence microscopy at a high acquisition rate of 10 frames/s. We use a Hidden Markov Model with two hidden states to (1) automatically identify directed migration segments of the tracks and (2) quantify the migration properties for comparison between states and between different experimental conditions. Comparing different cellular conditions, we show that the knockout of the peroxisomal membrane protein PEX14 leads to a decrease in the directed movement due to a lowered binding probability to the microtubule. However, it does not eradicate binding, highlighting further microtubule-binding mechanisms of peroxisomes than via PEX14 . In contrast, structural changes of the microtubular network explain perceived eradication of directed movement by disassembly of microtubules by Nocodazole-treatment.

A wide variety of treatments have been developed to improve respiratory function and quality of life in patients with bilateral vocal fold paresis (BVFP). One experimental method is the electrical activation of the posterior cricoarytenoid (PCA) muscle with a laryngeal pacemaker (LP) to open the vocal folds. We used an ovine (sheep) model of unilateral VFP to study the long-term effects of functional electrical stimulation on the PCA muscles. The left recurrent laryngeal nerve was cryo-damaged in all animals and an LP was implanted except for the controls. After a reinnervation phase of six months, animals were pooled into groups that received either no treatment, implantation of an LP only, or implantation of an LP and six months of stimulation with different duty cycles. Automated image analysis of fluorescently stained PCA cross-sections was performed to assess relevant muscle characteristics. We observed a fast-to-slow fibre type shift in response to nerve damage and stimulation, but no complete conversion to a slow-twitch-muscle. Fibre size, proportion of hybrid fibres, and intramuscular collagen content were not substantially altered by the stimulation. These results demonstrate that 30 Hz burst stimulation with duty cycles of 40% and 70% did not induce PCA atrophy or fibrosis. Thus, long-term stimulation with an LP is a promising approach for treating BVFP in humans without compromising muscle conditions.

Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities. Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus . IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Staphylococcus aureus colonizes epithelial surfaces, but it can also cause severe infections. The aim of this work was to investigate whether bacterial virulence correlates with defined types of tissue infections. For this, we collected 10–12 clinical S. aureus strains each from nasal colonization, and from patients with endoprosthesis infection, hematogenous osteomyelitis, and sepsis. All strains were characterized by genotypic analysis, and by the expression of virulence factors. The host–pathogen interaction was studied through several functional assays in osteoblast cultures. Additionally, selected strains were tested in a murine sepsis/osteomyelitis model. We did not find characteristic bacterial features for the defined infection types; rather, a wide range in all strain collections regarding cytotoxicity and invasiveness was observed. Interestingly, all strains were able to persist and to form small colony variants (SCVs). However, the low-cytotoxicity strains survived in higher numbers, and were less efficiently cleared by the host than the highly cytotoxic strains. In summary, our results indicate that not only destructive, but also low-cytotoxicity strains are able to induce infections. The low-cytotoxicity strains can successfully survive, and are less efficiently cleared from the host than the highly cytotoxic strains, which represent a source for chronic infections. The understanding of this interplay/evolution between the host and the pathogen during infection, with specific attention towards low-cytotoxicity isolates, will help to optimize treatment strategies for invasive and therapy-refractory infection courses.

Limited availability and side effects of opioids have led to an increased use of non-opioid analgesia in animal disease models. However, by affecting the immune-inflammatory reactions, analgesia may disrupt the resolution of the host inflammation and modulate the survival in septic animals. This study used a clinically relevant sepsis mouse model of peritoneal contamination and infection (PCI) to investigate the antinociceptive and anti-inflammatory properties of two non-opioid analgesics. Adult C57BL/6J mice were intraperitoneally injected with a human feces suspension and received either no analgesics (Non-A), Meloxicam, or Metamizole orally. The mice were monitored for pain and illness. Mortality was assessed at 7 days post-PCI. A separate group of mice was sacrificed 24 hours after infection. Blood, peritoneal lavage fluid (PLF), liver, and spleen were harvested for pathogen load quantification via qPCR, macrophage phenotyping, neutrophil infiltration/activation, and systemic/tissue cytokine release by flow cytometry. Meloxicam but not Metamizole reduced the mortality of septic mice by 31% on day 7 compared to the Non-A group. Both analgesics effectively alleviated pain but did not affect illness severity, body weight, and temperature. Meloxicam quadrupled the bacterial burden in the blood and PLF. In high IL-6 responders, Meloxicam treatment was associated with reduced circulating IL-10 and IL-1β compared to the Non-A septic group. In low IL-6 responders, Meloxicam increased circulating MCP-1 levels and decreased PGE2 levels compared to Non-A septic mice. Notably, Meloxicam reduced spleen neutrophil infiltration by 20% compared to two other sepsis groups. Metamizole and Meloxicam effectively relieved pain and increased the animals' basal activity in the PCI sepsis model. Meloxicam prolonged survival yet triggered maladaptive responses due to its immunosuppressive features that decreased tissue bacterial clearance during sepsis. In contrast, Metamizole constitutes a safe and effective non-opioid alternative for analgesic control in the non-surgical PCI sepsis model.

Application of personalized medicine requires integration of different data to determine each patient’s unique clinical constitution. The automated analysis of medical data is a growing field where different machine learning techniques are used to minimize the time-consuming task of manual analysis. The evaluation, and often training, of automated classifiers requires manually labelled data as ground truth. In many cases such labelling is not perfect, either because of the data being ambiguous even for a trained expert or because of mistakes. Here we investigated the interobserver variability of image data comprising fluorescently stained circulating tumor cells and its effect on the performance of two automated classifiers, a random forest and a support vector machine. We found that uncertainty in annotation between observers limited the performance of the automated classifiers, especially when it was included in the test set on which classifier performance was measured. The random forest classifier turned out to be resilient to uncertainty in the training data while the support vector machine’s performance is highly dependent on the amount of uncertainty in the training data. We finally introduced the consensus data set as a possible solution for evaluation of automated classifiers that minimizes the penalty of interobserver variability.

The receptor tyrosine kinase FLT3 is expressed in myeloid and lymphoid progenitor cells. Activating mutations in FLT3 occur in 25–30% of acute myeloid leukaemia (AML) patients. Most common are internal tandem duplications of sequence (ITD) leading to constitutive FLT3-ITD kinase activity with an altered signalling quality promoting leukaemic cell transformation. Here, we observed the attenuating role of the receptor-like protein tyrosine phosphatase (RPTP) CD45/Ptprc in FLT3 signalling in vivo. Low level expression of this abundant RPTP correlates with a poor prognosis of FLT3-ITD-positive AML patients. To get a further insight into the regulatory role of Ptprc in FLT3-ITD activity in vivo, Ptprc knock-out mice were bred with FLT3-ITD knock-in mice. Inactivation of the Ptprc gene in FLT3-ITD mice resulted in a drastically shortened life span and development of severe monocytosis, a block in B-cell development and anaemia. The myeloproliferative phenotype was associated with extramedullary haematopoiesis, splenohepatomegaly and severe alterations of organ structures. The phenotypic alterations were associated with increased transforming signalling of FLT3-ITD, including activation of its downstream target STAT5. These data reveal the capacity of Ptprc for the regulation of FLT3-ITD signalling activity in vivo. In addition, histopathology and computed tomography (CT) revealed an unexpected bone phenotype; the FLT3-ITD Ptprc -/- mice, but none of the controls, showed pronounced alterations in bone morphology and, in part, apparent features of osteoporosis. In the spleen, ectopic bone formation was observed. The observed bone phenotypes suggest a previously unappreciated capacity of FLT3-ITD (and presumably FLT3) to regulate bone development/remodelling, which is under negative control of CD45/Ptprc. Key points Low PTPRC expression of FLT3-ITD-positive AML patients correlates with poor prognosis FLT3-ITD/ Ptprc -/- mice develop severe monocytosis, a block in B-cell formation and anaemia FLT3-ITD/ Ptprc -/- mice develop myeloproliferative neoplasm with extramedullary haematopoiesis and splenohepatomegaly Inactivation of Ptprc in the presence of FLT3-ITD results in cortical porosity and ectopic bone formation Ptprc is negatively regulating transforming FLT3-ITD signalling in vivo

The assessment of bone damage is required to evaluate disease severity and treatment efficacy both in arthritis patients and in experimental arthritis models. Today there is still a lack of in vivo methods that enable the quantification of arthritic processes at an early stage of the disease. We performed longitudinal in vivo imaging with [ 18 F]-fluoride PET/CT before and after experimental arthritis onset for diseased and control DBA/1 mice and assessed arthritis progression by clinical scoring, tracer uptake studies and bone volume as well as surface roughness measurements. Arthritic animals showed significantly increased tracer uptake in the paws compared to non-diseased controls. Automated CT image analysis revealed increased bone surface roughness already in the earliest stage of the disease. Moreover, we observed clear differences between endosteal and periosteal sites of cortical bone regarding surface roughness. This study shows that in vivo PET/CT imaging is a favorable method to study arthritic processes, enabling the quantification of different aspects of the disease like pathological bone turnover and bone alteration. Especially the evaluation of bone surface roughness is sensitive to early pathological changes and can be applied to study the dynamics of bone erosion at different sites of the bones in an automated fashion.

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens.Key points• Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes.• These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages.• Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.