lipoteichoic acids are essential for pneumococcal colonization and membrane integrity.

INTRODUCTION: The hydrophilic, polymeric chain of the lipoteichoic acid (LTA) of the Gram-positive pathobiont Streptococcus pneumoniae is covalently linked to the glycosylglycerolipid -D-glucopyranosyl-(1,3)-diacylglycerol by the lipoteichoic acid ligase TacL, leading to its fixation in the cytoplasmic membrane. Pneumococcal LTA, sharing identical repeating units with the wall teichoic acids, are dispensable for normal growth but required for full-virulence in invasive infections. METHODS: Mutants deficient in TacL and complemented strains constructed were tested for their growth, resistance against oxidative stress and susceptibility against antimicrobial peptides. Further, the membrane fluidity of pneumococci, their capability to adhere to lung epithelial cells and virulence in a Galleria mellonella as well as intranasal mouse infection model were assessed. RESULTS: In the present study, we indicate that LTA is already indispensable for pneumococcal adherence to human nasopharyngeal cells and colonization in an intranasal mouse infection model. Mutants deficient for TacL did not show morphological defects. However, our analysis of pneumococcal membranes in different serotypes showed an altered membrane fluidity and surface protein abundance of lipoproteins in mutants deficient for LTA but not WTA. These mutants had a decreased membrane fluidity, exhibited higher amounts of lipoproteins, and showed an increased susceptibility to antimicrobial peptides. In complemented mutant strains this defect was fully restored. CONCLUSION: Taken together, LTA is crucial for colonization and required to effectively protect pneumococci from innate immune defence mechanisms by maintaining the membrane integrity.

Molecular and structural basis of oligopeptide recognition by the Ami transporter system in pneumococci.

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.

Hydrogen peroxide is responsible for the cytotoxic effects of Streptococcus pneumoniae on primary microglia in the absence of pneumolysin.

INTRODUCTION: Streptococcus pneumoniae is the most common cause of bacterial meningitis and meningoencephalitis in humans. The bacterium produces numerous virulence determinants, among them hydrogen peroxide (H2O2) and pneumolysin (Ply), which contribute to bacterial cytotoxicity. Microglia, the resident phagocytes in the brain, are distinct from other macrophages, and we thus compared their susceptibility to pneumococcal toxicity and their ability to phagocytose pneumococci with those of bone marrow-derived macrophages (BMDM). METHODS: Microglia and BMDM were co-incubated with S. pneumoniae D39 to analyze survival of phagocytes by fluorescence microscopy, bacterial growth by quantitative plating, and phagocytosis by an antibiotic protection assay. Ply was detected by hemolysis assay and Western blot analysis. RESULTS: We found that microglia were killed during pneumococcal infection with a wild-type and an isogenic ply-deficient mutant, whereas viability of BMDM was not affected by pneumococci. Treatment with recombinant Ply showed a dose-dependent cytotoxic effect on microglia and BMDM. However, high concentrations of recombinant Ply were required and under the chosen experimental conditions, Ply was not detectable in the supernatant during infection of microglia. Inactivation of H2O2 by exogenously added catalase abolished its cytotoxic effect. Consequently, infection of microglia with pneumococci deficient for the pyruvate oxidase SpxB, primarily producing H2O2, resulted in reduced killing of microglia. CONCLUSION: Taken together, in the absence of Ply, H2O2 caused cell death in primary phagocytes in concentrations produced by pneumococci.

The single D380 amino acid substitution increases pneumolysin cytotoxicity toward neuronal cells.

Bacterial meningitis, frequently caused by Streptococcus pneumoniae (pneumococcus), represents a substantial global health threat leading to long-term neurological disorders. This study focused on the cholesterol-binding toxin pneumolysin (PLY) released by pneumococci, specifically examining clinical isolates from patients with meningitis and comparing them to the PLY-reference S. pneumoniae strain D39. Clinical isolates exhibit enhanced PLY release, likely due to a significantly higher expression of the autolysin LytA. Notably, the same single amino acid (aa) D380 substitution in the PLY D4 domain present in all clinical isolates significantly enhances cholesterol binding, pore-forming activity, and cytotoxicity toward SH-SY5Y-derived neuronal cells. Scanning electron microscopy of human neuronal cells and patch clamp electrophysiological recordings on mouse brain slices confirm the enhanced neurotoxicity of the PLY variant carrying the single aa substitution. This study highlights how a single aa modification enormously alters PLY cytotoxic potential, emphasizing the importance of PLY as a major cause of the neurological sequelae associated with pneumococcal meningitis.

Using mobile eye tracking to measure cognitive load through gaze behavior during walking in lower limb prosthesis users: A preliminary assessment.

BACKGROUND: Lower limb amputation does not affect only physical and psychological functioning but the use of a prosthetic device can also lead to increased cognitive demands. Measuring cognitive load objectively is challenging, and therefore, most studies use questionnaires that are easy to apply but can suffer from subjective bias. Motivated by this, the present study investigated whether a mobile eye tracker can be used to objectively measure cognitive load by monitoring gaze behavior during a set of motor tasks. METHODS: Five prosthetic users and eight able-bodied controls participated in this study. Eye tracking data and kinematics were recorded during a set of motor tasks (level ground walking, walking on uneven terrain, obstacle avoidance, stairs up and ramp down, as well as ramp up and stairs down) while the participants were asked to focus their gaze on a visual target for as long as possible. Target fixation times and increase in pupil diameters were determined and correlated to subjective ratings of cognitive load. FINDINGS: Overall, target fixation time and pupil diameter showed strong negative and positive correlations, respectively, to the subjective rating of cognitive load in the able-bodied controls (-0.75 and 0.80, respectively). However, the individual correlation strength, and in some cases, even the sign, was different across participants. A similar trend could be observed in prosthetic users. INTERPRETATION: The results of this study showed that a mobile eye tracker may be used to estimate cognitive load in prosthesis users during locomotor tasks. This paves the way to establish a new approach to assessing cognitive load, which is objective and yet practical and simple to administer. Nevertheless, future studies should corroborate these results by comparing them to other objective measures as well as focus on translating the proposed approach outside of a laboratory.

Isoflurane, like sepsis, decreases CYP1A2 liver enzyme activity in intensive care patients: a clinical study and network model.

PURPOSE: Liver function of intensive care patients is routinely monitored by static blood pathology. For specific indications, liver specific cytochrome activity may be measured by the commercially available maximum liver function capacity (LiMAx) test via quantification of the cytochrome P450 1A2 (CYP1A2) dependent C-methacetin metabolism. Sedation with the volatile anesthetic isoflurane was suspected to abrogate the correlation of LiMAx test with global liver function. We hypothesized that isoflurane has a CYP1A2-activity and LiMAx test result decreasing effect. METHODS: In this monocentric, observational clinical study previously liver healthy intensive care patients, scheduled to be changed from propofol to isoflurane sedation, were enrolled. LiMAx testing was done before, during and after termination of isoflurane sedation. RESULTS: The mean LiMAx value decreased during isoflurane sedation. Septic patients (n = 11) exhibited lower LiMAx values compared to non-septic patients (n = 11) at all time points. LiMAx values decreased with isoflurane from 140 ± 82 to 30 ± 34 µg kg-1 h-1 in the septic group and from 253 ± 92 to 147 ± 131 µg kg-1 h-1 in the non-septic group while laboratory markers did not imply significant hepatic impairment. Lactate increased during isoflurane inhalation without clinical consequence. CONCLUSION: Sepsis and isoflurane have independently demonstrated an effect on reducing the hepatic CYP1A2-activity. A network model was constructed that could explain the mechanism through the influence of isoflurane on hypoxia inducible factor (HIF-1α) by upregulation of the hypoxia-inducible pathway and the downregulation of CYP1A2-activity via the ligand-inducible pathway. Thus, the increased anaerobic metabolism may result in lactate accumulation. The influence of isoflurane sedation on the validated correlation of global liver function with CYP1A2-activity measured by LiMAx testing needs to be investigated in more detail.

Assessing SOFA score trajectories in sepsis using machine learning: A pragmatic approach to improve the accuracy of mortality prediction.

INTRODUCTION: An increasing amount of longitudinal health data is available on critically ill septic patients in the age of digital medicine, including daily sequential organ failure assessment (SOFA) score measurements. Thus, the assessment in sepsis focuses increasingly on the evaluation of the individual disease's trajectory. Machine learning (ML) algorithms may provide a promising approach here to improve the evaluation of daily SOFA score dynamics. We tested whether ML algorithms can outperform the conventional ΔSOFA score regarding the accuracy of 30-day mortality prediction. METHODS: We used the multicentric SepsisDataNet.NRW study cohort that prospectively enrolled 252 sepsis patients between 03/2018 and 09/2019 for training ML algorithms, i.e. support vector machine (SVM) with polynomial kernel and artificial neural network (aNN). We used the Amsterdam UMC database covering 1,790 sepsis patients for external and independent validation. RESULTS: Both SVM (AUC 0.84; 95% CI: 0.71-0.96) and aNN (AUC 0.82; 95% CI: 0.69-0.95) assessing the SOFA scores of the first seven days led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score between day 1 and 7 (AUC 0.73; 95% CI: 0.65-0.80; p = 0.02 and p = 0.05, respectively). These differences were even more prominent the shorter the time interval considered. Using the SOFA scores of day 1 to 3 SVM (AUC 0.82; 95% CI: 0.68 0.95) and aNN (AUC 0.80; 95% CI: 0.660.93) led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score (AUC 0.66; 95% CI: 0.58-0.74; p < 0.01 and p < 0.01, respectively). Strikingly, all these findings could be confirmed in the independent external validation cohort. CONCLUSIONS: The ML-based algorithms using daily SOFA scores markedly improved the accuracy of mortality compared to the conventional ΔSOFA score. Therefore, this approach could provide a promising and automated approach to assess the individual disease trajectory in sepsis. These findings reflect the potential of incorporating ML algorithms as robust and generalizable support tools on intensive care units.

The Aquaporin 3 Polymorphism (rs17553719) Is Associated with Sepsis Survival and Correlated with IL-33 Secretion.

Sepsis is a common life-threatening disease caused by dysregulated immune response and metabolic acidosis which lead to organ failure. An abnormal expression of aquaporins plays an important role in organ failure. Additionally, genetic variants in aquaporins impact on the outcome in sepsis. Thus, we investigated the polymorphism (rs17553719) and expression of aquaporin-3 (AQP3) and correlated these measurements with the survival of sepsis patients. Accordingly, we collected blood samples on several days (plus clinical data) from 265 sepsis patients who stayed in different ICUs in Germany. Serum plasma, DNA, and RNA were then separated to detect the promotor genotypes of AQP3 mRNA expression of AQP3 and several cytokines. The results showed that the homozygote CC genotype exhibited a significant decrease in 30-day survival (38.9%) compared to the CT (66.15%) and TT genotypes (76.3%) (p = 0.003). Moreover, AQP3 mRNA expression was significantly higher and nearly doubled in the CC compared to the CT (p = 0.0044) and TT genotypes (p = 0.018) on the day of study inclusion. This was accompanied by an increased IL-33 concentration in the CC genotype (day 0: p = 0.0026 and day 3: p = 0.008). In summary, the C allele of the AQP3 polymorphism (rs17553719) shows an association with increased AQP3 expression and IL-33 concentration accompanied by decreased survival in patients with sepsis.

AQP3 and AQP9-Contrary Players in Sepsis?

Sepsis involves an immunological systemic response to a microbial pathogenic insult, leading to a cascade of interconnected biochemical, cellular, and organ-organ interaction networks. Potential drug targets can depict aquaporins, as they are involved in immunological processes. In immune cells, AQP3 and AQP9 are of special interest. In this study, we tested the hypothesis that these aquaporins are expressed in the blood cells of septic patients and impact sepsis survival. Clinical data, routine laboratory parameters, and blood samples from septic patients were analyzed on day 1 and day 8 after sepsis diagnosis. AQP expression and cytokine serum concentrations were measured. AQP3 mRNA expression increased over the duration of sepsis and was correlated with lymphocyte count. High AQP3 expression was associated with increased survival. In contrast, AQP9 expression was not altered during sepsis and was correlated with neutrophil count, and low levels of AQP9 were associated with increased survival. Furthermore, AQP9 expression was an independent risk factor for sepsis lethality. In conclusion, AQP3 and AQP9 may play contrary roles in the pathophysiology of sepsis, and these results suggest that AQP9 may be a novel drug target in sepsis and, concurrently, a valuable biomarker of the disease.

TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade.

IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.

Treatment of multiple-beam X-ray diffraction in energy-dependent measurements.

During X-ray diffraction experiments on single crystals, the diffracted beam intensities may be affected by multiple-beam X-ray diffraction (MBD). This effect is particularly frequent at higher X-ray energies and for larger unit cells. The appearance of this so-called Renninger effect often impairs the interpretation of diffracted intensities. This applies in particular to energy spectra analysed in resonant experiments, since during scans of the incident photon energy these conditions are necessarily met for specific X-ray energies. This effect can be addressed by carefully avoiding multiple-beam reflection conditions at a given X-ray energy and a given position in reciprocal space. However, areas which are (nearly) free of MBD are not always available. This article presents a universal concept of data acquisition and post-processing for resonant X-ray diffraction experiments. Our concept facilitates the reliable determination of kinematic (MBD-free) resonant diffraction intensities even at relatively high energies which, in turn, enables the study of higher absorption edges. This way, the applicability of resonant diffraction, e.g. to reveal the local atomic and electronic structure or chemical environment, is extended for a vast majority of crystalline materials. The potential of this approach compared with conventional data reduction is demonstrated by the measurements of the Ta L3 edge of well studied lithium tantalate LiTaO3.

The type-2 Streptococcus canis M protein SCM-2 binds fibrinogen and facilitates antiphagocytic properties.

Streptococcus canis is a zoonotic agent that causes severe invasive diseases in domestic animals and humans, but little is known about its pathogenesis and virulence mechanisms so far. SCM, the M-like protein expressed by S. canis, is considered one of the major virulence determinants. Here, we report on the two distinct groups of SCM. SCM-1 proteins were already described to interact with its ligands IgG and plasminogen as well as with itself and confer antiphagocytic capability of SCM-1 expressing bacterial isolates. In contrast, the function of SCM-2 type remained unclear to date. Using whole-genome sequencing and subsequent bioinformatics, FACS analysis, fluorescence microscopy and surface plasmon resonance spectrometry, we demonstrate that, although different in amino acid sequence, a selection of diverse SCM-2-type S. canis isolates, phylogenetically representing the full breadth of SCM-2 sequences, were able to bind fibrinogen. Using targeted mutagenesis of an SCM-2 isolate, we further demonstrated that this strain was significantly less able to survive in canine blood. With respect to similar studies showing a correlation between fibrinogen binding and survival in whole blood, we hypothesize that SCM-2 has an important contribution to the pathogenesis of S. canis in the host.

[Pulmonary hypertension associated with lung disease]. / Pulmonale Hypertonie assoziiert mit Lungenerkrankungen.

Lung diseases and hypoventilation syndromes are often associated with pulmonary hypertension (PH). In most cases, PH is not severe. This is defined hemodynamically by a mean pulmonary arterial pressure (PAPm) > 20 mmHg, a pulmonary arterial wedge pressure (PAWP) ≤ 15 mmHg and a pulmonary vascular resistance of ≤ 5 Wood units (WU). Both the non-severe (PVR ≤ 5 WU) and much more the severe PH (PVR > 5 WU) have an unfavorable prognosis.If PH is suspected, it is recommended to primarily check whether risk factors for pulmonary arterial hypertension (PAH, group 1 PH) or chronic thromboembolic pulmonary hypertension (CTEPH, group 4 PH) are present. If risk factors are present or there is a suspicion of severe PH in lung patients, it is recommended that the patient should be presented to a PH outpatient clinic promptly.For patients with severe PH associated with lung diseases, personalized, individual therapy is recommended - if possible within the framework of therapy studies. Currently, a therapy attempt with PH specific drugs should only be considered in COPD patients if the associated PH is severe and a "pulmonary vascular" phenotype (severe precapillary PH, but typically only mild to moderate airway obstruction, no or mild hypercapnia and DLCO < 45 % of predicted value) is present. In patients with severe PH associated with interstitial lung disease phosphodiesterase-5-inhibitors may be considered in individual cases. Inhaled treprostinil may be considered also in non-severe PH in this patient population.

Methylated Septin9 identified patients with colorectal carcinoma and showed higher sensitivity than conventional biomarkers in detecting tumor.

INTRODUCTION: It is worth noting the limitations in sensitivity of the existing biomarkers carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) in detection of colorectal cancer (CRC). In our study, we address the performance of the liquid biopsy biomarker "methylated septin 9" (mSEPT9) in the detection and disease surveillance of CRC. MATERIALS AND METHODS: The monocentric prospective survey encompassed 120 patients diagnosed with CRC who underwent planned curative resection between December 2018 and December 2020. Blood samples were collected from the participants preoperatively as well as at 7 days, 6 weeks, and 3 months postoperatively. The presence of mSEPT9, CEA, and CA 19-9 was detected using the pro Epi Colon® 2.0 CE test, Elecsys® CEA, and Elecsys® CA19-9 electrochemiluminescence immunoassay, respectively. RESULTS: In the preoperative setting, mSEPT9 demonstrated superior capability in identifying patients with CRC compared to CEA and CA 19-9, with detection rates of 57%, 32%, and 18% respectively. Combining all three biomarkers increased the overall sensitivity to 66% preoperatively. In considering UICC stage and T-status, mSEPT9 exhibited higher sensitivity across all stages in comparison with conventional tumor markers, and 65% of patients with metastases were identified postoperatively through mSEPT9. Tumor recognition after surgery was achieved with the sensitivity of 72% and specificity of 91%. CONCLUSIONS: We recommend using mSEPT9 as a non-invasive diagnostic tool for the ongoing monitoring of patients with CRC. The sensitivity and specificity exhibited by mSEPT9 in recognition of tumor after surgery, highlights its particular potential for monitoring of CRC patients.

The choline-binding proteins PspA, PspC, and LytA of Streptococcus pneumoniae and their interaction with human endothelial and red blood cells.

Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that can colonize the upper respiratory tract. It is a leading cause of a wide range of infectious diseases, including community-acquired pneumonia and meningitis. Pneumococcal infections cause 1-2 million deaths per year, most of which occur in developing countries. Here, we focused on three choline-binding proteins (CBPs), i.e., PspC, PspA, and LytA. These pneumococcal proteins have different surface-exposed regions but share related choline-binding anchors. These surface-exposed pneumococcal proteins are in direct contact with host cells and have diverse functions. We explored the role of the three CBPs on adhesion and pathogenicity in a human host by performing relevant imaging and functional analyses, such as electron microscopy, confocal laser scanning microscopy, and functional quantitative assays, targeting biofilm formation and the hemolytic capacity of S. pneumoniae. In vitro biofilm formation assays and electron microscopy experiments were used to examine the ability of knockout mutant strains lacking the lytA, pspC, or pspA genes to adhere to surfaces. We found that LytA plays an important role in robust synthesis of the biofilm matrix. PspA and PspC appeared crucial for the hemolytic effects of S. pneumoniae on human red blood cells. Furthermore, all knockout mutants caused less damage to endothelial cells than wild-type bacteria, highlighting the significance of each CPB for the overall pathogenicity of S. pneumoniae. Hence, in addition to their structural function within the cell wall of S. pneumoniae, each of these three surface-exposed CBPs controls or mediates multiple steps during bacterial pathogenesis.

Empowering Researchers to Query Medical Data and Biospecimens by Ensuring Appropriate Usability of a Feasibility Tool: Evaluation Study.

BACKGROUND: The Aligning Biobanking and Data Integration Centers Efficiently project aims to harmonize technologies and governance structures of German university hospitals and their biobanks to facilitate searching for patient data and biospecimens. The central element will be a feasibility tool for researchers to query the availability of samples and data to determine the feasibility of their study project. OBJECTIVE: The objectives of the study were as follows: an evaluation of the overall user interface usability of the feasibility tool, the identification of critical usability issues, comprehensibility of the underlying ontology operability, and analysis of user feedback on additional functionalities. From these, recommendations for quality-of-use optimization, focusing on more intuitive usability, were derived. METHODS: To achieve the study goal, an exploratory usability test consisting of 2 main parts was conducted. In the first part, the thinking aloud method (test participants express their thoughts aloud throughout their use of the tool) was complemented by a quantitative questionnaire. In the second part, the interview method was combined with supplementary mock-ups to collect users' opinions on possible additional features. RESULTS: The study cohort rated global usability of the feasibility tool based on the System Usability Scale with a good score of 81.25. The tasks assigned posed certain challenges. No participant was able to solve all tasks correctly. A detailed analysis showed that this was mostly because of minor issues. This impression was confirmed by the recorded statements, which described the tool as intuitive and user friendly. The feedback also provided useful insights regarding which critical usability problems occur and need to be addressed promptly. CONCLUSIONS: The findings indicate that the prototype of the Aligning Biobanking and Data Integration Centers Efficiently feasibility tool is headed in the right direction. Nevertheless, we see potential for optimization primarily in the display of the search functions, the unambiguous distinguishability of criteria, and the visibility of their associated classification system. Overall, it can be stated that the combination of different tools used to evaluate the feasibility tool provided a comprehensive picture of its usability.

Blocking P2Y2 purinergic receptor prevents the development of lipopolysaccharide-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality resulting from a direct or indirect injury of the lung. It is characterized by a rapid alveolar injury, lung inflammation with neutrophil accumulation, elevated permeability of the microvascular-barrier leading to an aggregation of protein-rich fluid in the lungs, followed by impaired oxygenation in the arteries and eventual respiratory failure. Very recently, we have shown an involvement of the Gq-coupled P2Y2 purinergic receptor (P2RY2) in allergic airway inflammation (AAI). In the current study, we aimed to elucidate the contribution of the P2RY2 in lipopolysaccharide (LPS)-induced ARDS mouse model. We found that the expression of P2ry2 in neutrophils, macrophages and lung tissue from animals with LPS-induced ARDS was strongly upregulated at mRNA level. In addition, ATP-neutralization by apyrase in vivo markedly attenuated inflammation and blocking of P2RY2 by non-selective antagonist suramin partially decreased inflammation. This was indicated by a reduction in the number of neutrophils, concentration of proinflammatory cytokines in the BALF, microvascular plasma leakage and reduced features of inflammation in histological analysis of the lung. P2RY2 blocking has also attenuated polymorphonuclear neutrophil (PMN) migration into the interstitium of the lungs in ARDS mouse model. Consistently, treatment of P2ry2 deficient mice with LPS lead to an amelioration of the inflammatory response showed by reduced number of neutrophils and concentrations of proinflammatory cytokines. In attempts to identify the cell type specific role of P2RY2, a series of experiments with conditional P2ry2 knockout animals were performed. We observed that P2ry2 expression in neutrophils, but not in the airway epithelial cells or CD4+ cells, was associated with the inflammatory features caused by ARDS. Altogether, our findings imply for the first time that increased endogenous ATP concentration via activation of P2RY2 is related to the pathogenesis of LPS-induced lung inflammation and may represent a potential therapeutic target for the treatment of ARDS and predictably assess new treatments in ARDS.

[Complaints and clinical findings six months after COVID-19: outpatient follow-up at the University Medical Center Freiburg]. / Persistierende Beschwerden 6 Monate nach COVID-19 ­ Erfahrungen aus der COVID-19-Nachsorgeambulanz des Universitätsklinikums Freiburg.

BACKGROUND: Increasing evidence suggests that some patients suffer from persistent symptoms for months after recovery from acute COVID-19. However, the clinical phenotype and its pathogenesis remain unclear. We here present data on complaints and results of a diagnostic workup of patients presenting to the post-COVID clinic at the University Medical Center Freiburg. METHODS: Retrospective data analysis of persistently symptomatic patients presenting to our clinic at least 6 months after onset of acute COVID-19. All patients were assessed by a doctor and routine laboratory analysis was carried out. Quality of life was assessed using SF-36 questionnaire. In case of specific persisting symptoms, further organ-specific diagnostic evaluation was performed, and patients were referred to respective departments/specialists. FINDINGS: 132 Patients (58 male, 74 female; mean age 53.8 years) presented to our clinic at least 6 months after COVID-19. 79 (60 %) had been treated as outpatients and 53 (40 %) as inpatients. Most common complaints were persistent fatigue (82 %) and dyspnea on exertion (61 %). Further common complaints were impairments of concentration (54 %), insomnia (43 %), and impairments of smell or taste (35 %). Quality of life was reduced in all sections of the SF-36 questionnaire, yielding a reduced working capacity. Significant pathological findings in laboratory, echocardiographic and radiological work-up were rare. Impairments in lung function tests were more common in previously hospitalized patients. CONCLUSION: Patients presenting 6 months after onset of acute COVID-19 suffer from a diverse spectrum of symptoms with impaired quality of life, also referred to as Long COVID or Post-Acute Sequelae of SARS-CoV-2 infection (PASC). Further research is needed to determine the frequency of these post-COVID syndromes and their pathogenesis, natural course and treatment options. Evaluation and management should be multi-disciplinary.

Immunomodulation by Hemoadsorption-Changes in Hepatic Biotransformation Capacity in Sepsis and Septic Shock: A Prospective Study.

BACKGROUND: Sepsis is often associated with liver dysfunction, which is an indicator of poor outcomes. Specific diagnostic tools that detect hepatic dysfunction in its early stages are scarce. So far, the immune modulatory effects of hemoadsorption with CytoSorb® on liver function are unclear. METHOD: We assessed the hepatic function by using the dynamic LiMAx® test and biochemical parameters in 21 patients with sepsis or septic shock receiving CytoSorb® in a prospective, observational study. Points of measurement: T1: diagnosis of sepsis or septic shock; T2 and T3: 24 h and 48 h after the start of CytoSorb®; T4: 24 h after termination of CytoSorb®. RESULTS: The hepatic biotransformation capacity measured by LiMAx® was severely impaired in up to 95 % of patients. Despite a rapid shock reversal under CytoSorb®, a significant improvement in LiMAx® values appeared from T3 to T4. This decline and recovery of liver function were not reflected by common parameters of hepatic metabolism that remained mostly within the normal range. CONCLUSIONS: Hepatic dysfunction can effectively and safely be diagnosed with LiMAx® in ventilated ICU patients under CytoSorb®. Various static liver parameters are of limited use since they do not adequately reflect hepatic dysfunction and impaired hepatic metabolism.