Influence of Kidney Environment Parameters on Antibiotic Efficacy Against Uropathogenic Escherichia coli.

BACKGROUND AND OBJECTIVE: Urinary tract infections (UTIs) are common infections affecting the urinary system, predominantly caused by bacterial pathogens, with Escherichia coli being the most frequent pathogen. Infections of the kidney (eg, pyelonephritis) are severe and challenging to treat, due to the specific tissue microenvironment. In this study, the influence of different parameters mimicking the kidney environment on the effectiveness of antibiotics prescribed for pyelonephritis on the growth of uropathogenic strains was analyzed. METHODS: To investigate the influence of different factors mimicking the kidney environment, we tested the effect of different kidney-representative concentrations of sodium chloride and urea, and different pH values on the efficacy of ertapenem, levofloxacin, and ceftriaxone. The effectiveness was assessed by determining the minimal inhibitory concentrations (MICs) against various E. coli strains. KEY FINDINGS AND LIMITATIONS: The study revealed that pH significantly influences the MIC values of levofloxacin. Acidification of the pH led to an increase of the MIC values, while an alkaline pH had the opposite effect. The influence of sodium chloride and urea concentrations was strain and antibiotic specific. Since three different antibiotics were tested in this study, further research with additional antibiotics is warranted. CONCLUSIONS AND CLINICAL IMPLICATIONS: These results suggest that the physicochemical conditions within the kidney can substantially influence the success of antibiotic therapy for pyelonephritis. Therefore, it is crucial for clinicians to consider these factors when selecting and dosing antibiotics. Further research is needed to evaluate a broader range of antibiotics and additional environmental parameters, to develop a more comprehensive understanding of how the kidney environment affects antimicrobial activity. This knowledge will be vital in optimizing treatment strategies for pyelonephritis, ultimately improving patient outcomes. PATIENT SUMMARY: The physicochemical conditions within the kidney influence the success of antibiotic therapy for pyelonephritis. Our findings are vital in optimizing treatment strategies and will ultimately improve patient outcomes.

Co-option of mitochondrial nucleic acid sensing pathways by HSV-1 UL12.5 for reactivation from latent Infection.

Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.

Canonical and Noncanonical Contribution of Thyroid Hormone Receptor Isoforms Alpha and Beta to Cardiac Hypertrophy and Heart Rate in Male Mice.

Background: Stimulation of ventricular hypertrophy and heart rate are two major cardiac effects of thyroid hormone (TH). The aim of this study was to determine in vivo which TH receptor (TR)-α or ß-and which mode of TR action-canonical gene expression or DNA-binding independent noncanonical action-mediate these effects. Methods: We compared global TRα and TRß knockout mice (TRαKO; TRßKO) with wild-type (WT) mice to determine the TR isoform responsible for T3 effects. The relevance of TR DNA binding was studied in mice with a mutation in the DNA-binding domain that selectively abrogates DNA binding and canonical TR action (TRαGS; TRßGS). Hearts were studied with echocardiography at baseline and after 7 weeks of T3 treatment. Gene expression was measured with real-time polymerase chain reaction. Heart rate was recorded with radiotelemetry transmitters for 7 weeks in untreated, hypothyroid, and T3-treated mice. Results: T3 induced ventricular hypertrophy in WT and TRßKO mice, but not in TRαKO mice. Hypertrophy was also induced in TRαGS mice. Thus, hypertrophy is mostly mediated by noncanonical TRα action. Similarly, repression of Mhy7 occurred in WT and TRαGS mice. Basal heart rate was largely dependent on canonical TRα action. But responsiveness to hypothyroidism and T3 treatment as well as expression of pacemaker gene Hcn2 were still preserved in TRαKO mice, demonstrating that TRß could compensate for absence of TRα. Conclusions: T3-induced cardiac hypertrophy could be attributed to noncanonical TRα action, whereas heart rate regulation was mediated by canonical TRα action. TRß could substitute for canonical but not noncanonical TRα action.

Scientific Advances in Understanding the Pathogenesis, Diagnosis, and Prevention of Urinary Tract Infection in the Past 10 Years.

Urinary tract infection (UTI) is a very common disease that is accompanied by various complications in the affected person. UTI triggers diverse inflammatory reactions locally in the infected urinary bladder and kidney, causing tissue destruction and organ failure. Moreover, systemic responses in the entire body carry the risk of urosepsis with far-reaching consequences. Understanding the cell-, organ-, and systemic mechanisms in UTI are crucial for prevention, early intervention, and current therapeutic approaches. This review summarizes the scientific advances over the last 10 years concerning pathogenesis, prevention, rapid diagnosis, and new treatment approaches. We also highlight the impact of the immune system and potential new therapies to reduce progressive and recurrent UTI.

The global burden of antimicrobial resistance - urinary tract infections.

Antimicrobial resistance (AMR) has emerged as a significant global healthcare problem. Antibiotic use has accelerated the physiologic process of AMR, particularly in Gram-negative pathogens. Urinary tract infections (UTIs) are predominantly of a Gram-negative nature. Uropathogens are evolutionarily highly adapted and selected strains with specific virulence factors, suggesting common mechanisms in how bacterial cells acquire virulence and AMR factors. The simultaneous increase in resistance and virulence is a complex and context-dependent phenomenon. Among known AMR mechanisms, the plenitude of different ß-lactamases is especially prominent. The risk for AMR in UTIs varies in different patient populations. A history of antibiotic consumption and the physiology of urinary flow are major factors that shape AMR prevalence. The urinary tract is in close crosstalk with the microbiome of other compartments, including the gut and genital tracts. In addition, pharmacokinetic properties and the physiochemical composition of urinary compartments can contribute to the emergence of AMR. Alternatives to antibiotic treatment and a broader approach to address bacterial infections are needed. Among the various alternatives studied, antimicrobial peptides and bacteriophage treatment appear to be highly promising approaches. We herein summarize the present knowledge of clinical and microbiological AMR in UTIs and discuss innovative approaches, namely new risk prediction tools and the use of non-antibiotic approaches to defend against uropathogenic microbes.

Proteome Analysis of Thyroid Hormone Transporter Mct8/Oatp1c1-Deficient Mice Reveals Novel Dysregulated Target Molecules Involved in Locomotor Function.

Thyroid hormone (TH) transporter MCT8 deficiency causes severe locomotor disabilities likely due to insufficient TH transport across brain barriers and, consequently, compromised neural TH action. As an established animal model for this disease, Mct8/Oatp1c1 double knockout (DKO) mice exhibit strong central TH deprivation, locomotor impairments and similar histo-morphological features as seen in MCT8 patients. The pathways that cause these neuro-motor symptoms are poorly understood. In this paper, we performed proteome analysis of brain sections comprising cortical and striatal areas of 21-day-old WT and DKO mice. We detected over 2900 proteins by liquid chromatography mass spectrometry, 67 of which were significantly different between the genotypes. The comparison of the proteomic and published RNA-sequencing data showed a significant overlap between alterations in both datasets. In line with previous observations, DKO animals exhibited decreased myelin-associated protein expression and altered protein levels of well-established neuronal TH-regulated targets. As one intriguing new candidate, we unraveled and confirmed the reduced protein and mRNA expression of Pde10a, a striatal enzyme critically involved in dopamine receptor signaling, in DKO mice. As altered PDE10A activities are linked to dystonia, reduced basal ganglia PDE10A expression may represent a key pathogenic pathway underlying human MCT8 deficiency.

Comparative transcriptomic and proteomic signature of lung alveolar macrophages reveals the integrin CD11b as a regulatory hub during pneumococcal pneumonia infection.

Introduction: Streptococcus pneumoniae is one of the main causes of community-acquired infections in the lung alveoli in children and the elderly. Alveolar macrophages (AM) patrol alveoli in homeostasis and under infectious conditions. However, the molecular adaptations of AM upon infections with Streptococcus pneumoniae are incompletely resolved. Methods: We used a comparative transcriptomic and proteomic approach to provide novel insights into the cellular mechanism that changes the molecular signature of AM during lung infections. Using a tandem mass spectrometry approach to murine cell-sorted AM, we revealed significant proteomic changes upon lung infection with Streptococcus pneumoniae. Results: AM showed a strong neutrophil-associated proteomic signature, such as expression of CD11b, MPO, neutrophil gelatinases, and elastases, which was associated with phagocytosis of recruited neutrophils. Transcriptomic analysis indicated intrinsic expression of CD11b by AM. Moreover, comparative transcriptomic and proteomic profiling identified CD11b as the central molecular hub in AM, which influenced neutrophil recruitment, activation, and migration. Discussion: In conclusion, our study provides novel insights into the intrinsic molecular adaptations of AM upon lung infection with Streptococcus pneumoniae and reveals profound alterations critical for effective antimicrobial immunity.

Correlation of tumor PD-L1 expression in different tissue types and outcome of PD-1-based immunotherapy in metastatic melanoma - analysis of the DeCOG prospective multicenter cohort study ADOREG/TRIM.

BACKGROUND: PD-1-based immune checkpoint inhibition (ICI) is the major backbone of current melanoma therapy. Tumor PD-L1 expression represents one of few biomarkers predicting ICI therapy outcome. The objective of the present study was to systematically investigate whether the type of tumor tissue examined for PD-L1 expression has an impact on the correlation with ICI therapy outcome. METHODS: Pre-treatment tumor tissue was collected within the prospective DeCOG cohort study ADOREG/TRIM (CA209-578; NCT05750511) between February 2014 and May 2020 from 448 consecutive patients who received PD-1-based ICI for non-resectable metastatic melanoma. The primary study endpoint was best overall response (BOR), secondary endpoints were progression-free (PFS) and overall survival (OS). All endpoints were correlated with tumor PD-L1 expression (quantified with clone 28-8; cutoff ≥5%) and stratified by tissue type. FINDINGS: Tumor PD-L1 was determined in 95 primary tumors (PT; 36.8% positivity), 153 skin/subcutaneous (34.0% positivity), 115 lymph node (LN; 50.4% positivity), and 85 organ (40.8% positivity) metastases. Tumor PD-L1 correlated with BOR if determined in LN (OR = 0.319; 95% CI = 0.138-0.762; P = 0.010), but not in skin/subcutaneous metastases (OR = 0.656; 95% CI = 0.311-1.341; P = 0.26). PD-L1 positivity determined on LN metastases was associated with favorable survival (PFS, HR = 0.490; 95% CI = 0.310-0.775; P = 0.002; OS, HR = 0.519; 95% CI = 0.307-0.880; P = 0.014). PD-L1 positivity determined in PT (PFS, HR = 0.757; 95% CI = 0.467-1.226; P = 0.27; OS; HR = 0.528; 95% CI = 0.305-0.913; P = 0.032) was correlated with survival to a lesser extent. No relevant survival differences were detected by PD-L1 determined in skin/subcutaneous metastases (PFS, HR = 0.825; 95% CI = 0.555-1.226; P = 0.35; OS, HR = 1.083; 95% CI = 0.698-1.681; P = 0.72). INTERPRETATION: For PD-1-based immunotherapy in melanoma, tumor PD-L1 determined in LN metastases was stronger correlated with therapy outcome than that assessed in PT or organ metastases. PD-L1 determined in skin/subcutaneous metastases showed no outcome correlation and therefore should be used with caution for clinical decision making. FUNDING: Bristol-Myers Squibb (ADOREG/TRIM, NCT05750511); German Research Foundation (DFG; Clinician Scientist Program UMEA); Else Kröner-Fresenius-Stiftung (EKFS; Medical Scientist Academy UMESciA).

Interleukin 17 signaling supports clinical benefit of dual CTLA-4 and PD-1 checkpoint inhibition in melanoma.

Recent studies suggest that BRAFV600-mutated melanomas in particular respond to dual anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibition (ICI). Here we identified an over-representation of interleukin (IL)-17-type 17 helper T (TH17) gene expression signatures (GES) in BRAFV600-mutated tumors. Moreover, high baseline IL-17 GES consistently predicted clinical responses in dual-ICI-treated patient cohorts but not in mono anti-CTLA-4 or anti-PD-1 ICI cohorts. High IL-17 GES corresponded to tumor infiltration with T cells and neutrophils. Accordingly, high neutrophil infiltration correlated with clinical response specifically to dual ICI, and tumor-associated neutrophils also showed strong IL-17-TH17 pathway activity and T cell activation capacity. Both the blockade of IL-17A and the depletion of neutrophils impaired dual-ICI response and decreased T cell activation. Finally, high IL-17A levels in the blood of patients with melanoma indicated a higher global TH17 cytokine profile preceding clinical response to dual ICI but not to anti-PD-1 monotherapy, suggesting a future role as a biomarker for patient stratification.

Dose-dependent effect of GFI1 expression in the reconstitution and the differentiation capacity of HSCs.

GFI1 is a transcriptional repressor and plays a pivotal role in regulating the differentiation of hematopoietic stem cells (HSCs) towards myeloid and lymphoid cells. Serial transplantation of Gfi1 deficient HSCs repopulated whole hematopoietic system but in a competitive setting involving wild-type HSCs, they lose this ability. The underlying mechanisms to this end are poorly understood. To better understand this, we used different mouse strains that express either loss of both Gfi1 alleles (Gfi1-KO), with reduced expression of GFI1 (GFI1-KD) or wild-type Gfi1/GFI1 (Gfi1-/GFI1-WT; corresponding to the mouse and human alleles). We observed that loss of Gfi1 or reduced expression of GFI1 led to a two to four fold lower number of HSCs (defined as Lin-Sca1+c-Kit+CD150+CD48-) compared to GFI1-WT mice. To study the functional influence of different levels of GFI1 expression on HSCs function, HSCs from Gfi1-WT (expressing CD45.1 + surface antigens) and HSCs from GFI1-KD or -KO (expressing CD45.2 + surface antigens) mice were sorted and co-transplanted into lethally irradiated host mice. Every 4 weeks, CD45.1+ and CD45.2 + on different lineage mature cells were analyzed by flow cytometry. At least 16 weeks later, mice were sacrificed, and the percentage of HSCs and progenitors including GMPs, CMPs and MEPs in the total bone marrow cells was calculated as well as their CD45.1 and CD45.2 expression. In the case of co-transplantation of GFI1-KD with Gfi1-WT HSCs, the majority of HSCs (81% ± 6%) as well as the majority of mature cells (88% ± 10%) originated from CD45.2 + GFI1-KD HSCs. In the case of co-transplantation of Gfi1-KO HSCs with Gfi1-WT HSCs, the majority of HSCs originated from CD45.2+ and therefore from Gfi1-KO (61% ± 20%); however, only a small fraction of progenitors and mature cells originated from Gfi1-KO HSCs (<1%). We therefore in summary propose that GFI1 has a dose-dependent role in the self-renewal and differentiation of HSCs.

Rapid and fully automated blood vasculature analysis in 3D light-sheet image volumes of different organs.

Light-sheet fluorescence microscopy (LSFM) can produce high-resolution tomograms of tissue vasculature with high accuracy. However, data processing and analysis is laborious due to the size of the datasets. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is ∼100 times faster than other existing vessel analysis tools, requires no user interaction, and integrates batch processing and parallelization. Employing an innovative dual Frangi filter approach, we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.

Acute effects of an exercise based multimodal in-patient rehabilitation protocol in male knee osteoarthritis patients and the two years follow-up sustainability.

BACKGROUND: Working with lifting and carrying heavy loads and kneeling postures with crawling, squats or heel seat position lead to progressive cartilage wear with premature degenerative changes. OBJECTIVE: To investigate the effects of the exercise based multimodal 'knee college' and its sustainability in patients with knee osteoarthritis with data assessments before and after a starter course, before a 1-year and a 2-year follow-up refresher course in a retrospective observational study. METHODS: A sample of 401 male patients (ICD10: M17 [arthrosis of knee]/ICF: s75011 [knee joint]) from the construction industries were assessed with Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), EuroQol (EQ-5D), Performance Assessment Capacity Testing (PACT), Isokinetic torque H/Q ratio and Physical Work Capacity Test (PWC). Retrospectively, after two years they were divided into three groups based on their intermediate sporting activity: gym (n = 194, age: 50.8±7.0, BMI: 28.8±4,3), home training (n = 110, age: 50.2±7.0, BMI: 28.4±4,2), no exercising (n = 97, age: 48.2±7.0, BMI: 29.2±4,6). RESULTS: Patients did not differ significantly in their demographic and anthropometric data prior to the rehab program. Significant interaction effects indicated group-dependent differing sustainability effects for the 2-year follow-up (all outcomes: p < 0.001, except for H/Q ratio: p = 0.03). Group-wise analyses revealed significant acute improvements (after 3-week in-patient starter rehab program: p < 0.05) for all groups in almost all outcomes (except the 'no sport' group, H/Q ratio p = 0.08). These effects remained significant (p < 0.001) only for the 'gym' group during the 1-year and 2-year follow-up. CONCLUSION: Our data indicate that 2-year sustainability of acute rehabilitation starter effects was demonstrated especially for patients with adherence and compliance to long-term gym based exercises.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

The interplay of thyroid hormones and the immune system - where we stand and why we need to know about it.

Over the past few years, growing evidence suggests direct crosstalk between thyroid hormones (THs) and the immune system. Components of the immune system were proposed to interfere with the central regulation of systemic TH levels. Conversely, THs regulate innate and adaptive immune responses as immune cells are direct target cells of THs. Accordingly, they express different components of local TH action, such as TH transporters or receptors, but our picture of the interplay between THs and the immune system is still incomplete. This review provides a critical overview of current knowledge regarding the interaction of THs and the immune system with the main focus on local TH action within major innate and adaptive immune cell subsets. Thereby, this review aims to highlight open issues which might help to infer the clinical relevance of THs in host defence in the context of different types of diseases such as infection, ischemic organ injury or cancer.

Going green: Development of a sustainable lipid-based enteric coating formulation for low-dose aspirin multiparticulate systems.

There is a rising awareness of pharmaceutical industry of both patient-centric and sustainable product development. Manufacturing of multiparticulate systems (MPS) with functional coating via solvent-free hot melt coating (HMC) can fulfill both requirements. An innovative lipid-based formulation was developed with the composition of palmitic acid and Grindsted® citrem BC-FS (BC-FS) for enteric coating of acetylsalicylic acid (ASA). The ASA crystals were directly hot melt coated to produce user-friendly low-dose ASA MPS for thromboembolism prophylaxis. Prior to HMC, rational boundaries for the process temperature were defined based on the melting and crystallization behavior of coating blend. Stability of coating in terms of resistance to heat stress and solidstate stability were screened via Fourier-transform infrared spectroscopy and x-ray diffraction. Exposure of coating blend to 100 °C for two hours did not cause any chemical degradation. Crystal growth of palmitic acid and polymorphic transformation in BC-FS were observed after storage under accelerated conditions, however did not significantly affect the ASA release from coating. The developed formulation is a unique solvent-free, lipid-based enteric composition and paves the way for sustainable green pharmaceutical manufacturing.

Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression.

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8+ T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

Eosinophils Suppress the Migration of T Cells Into the Brain of Plasmodium berghei-Infected Ifnar1-/- Mice and Protect Them From Experimental Cerebral Malaria.

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.

Digital Quantification of Tumor PD-L1 Predicts Outcome of PD-1-Based Immune Checkpoint Therapy in Metastatic Melanoma.

BACKGROUND: PD-1-based immune checkpoint blockade (ICB) is a highly effective therapy in metastatic melanoma. However, 40-60% of patients are primarily resistant, with valid predictive biomarkers currently missing. This study investigated the digitally quantified tumor PD-L1 expression for ICB therapy outcome prediction. PATIENTS AND METHODS: Tumor tissues taken prior to PD-1-based ICB for unresectable metastatic disease were collected within the prospective multicenter Tissue Registry in Melanoma (TRIM). PD-L1 expression (clone 28-8; cut-off=5%) was determined by digital and physician quantification, and correlated with therapy outcome (best overall response, BOR; progression-free survival, PFS; overall survival, OS). RESULTS: Tissue samples from 156 patients were analyzed (anti-PD-1, n=115; anti-CTLA-4+anti-PD-1, n=41). Patients with PD-L1-positive tumors showed an improved response compared to patients with PD-L1-negative tumors, by digital (BOR 50.5% versus 32.2%; p=0.026) and physician (BOR 54.2% versus 36.6%; p=0.032) quantification. Tumor PD-L1 positivity was associated with a prolonged PFS and OS by either digital (PFS, 9.9 versus 4.6 months, p=0.021; OS, not reached versus 13.0 months, p=0.001) or physician (PFS, 10.6 versus 5.6 months, p=0.051; OS, not reached versus 15.6 months, p=0.011) quantification. Multivariable Cox regression revealed digital (PFS, HR=0.57, p=0.007; OS, HR=0.44, p=0.001) and physician (OS, HR=0.54, p=0.016) PD-L1 quantification as independent predictors of survival upon PD-1-based ICB. The combination of both methods identified a patient subgroup with particularly favorable therapy outcome (PFS, HR=0.53, p=0.011; OS, HR=0.47, p=0.008). CONCLUSION: Pre-treatment tumor PD-L1 positivity predicted a favorable outcome of PD-1-based ICB in melanoma. Herein, digital quantification was not inferior to physician quantification, and should be further validated for clinical use.

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome.

Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.