Background The tumor immune microenvironment (TIME) of canine and feline mammary carcinomas (CMC and FMC) comprises a complex collection of immune cells that remains underinvestigated. We performed extensive immunohistochemical characterization with specific immune cell markers on retrospectively collected grade I, II and III CMC and FMC to spatially and quantitatively investigate theTIME-associated changes in correlation with the tumor grade, tumor size and necrosis for both species. Results Antigen-presenting cells, tumor-associated macrophages and-neutrophils, were mainly located in the tumoral stroma and formed an immunosuppressive border surrounding the tumor core, most prominently in grade III carcinomas. Activated T lymphocytes were either scattered or clustered in B lymphocyte-rich aggregates in the tumoral stroma, while infiltrated T lymphocytes in the tumor core showed no activation. Grade III mammary carcinomas were characterized by more abundant vascularization compared to grade I/II CMC and FMC. Tumor size, but not necrosis, was identified as influencing factor for both immune cell infiltration and blood vessel density in CMC and FMC. Conclusion Collectively, these data indicate that grade III CMC and FMC are associated with an immunosuppressed, vascularized and proliferative TIME, warranting further exploration of the underlying pro-tumorigenic mechanism for novel prognostic and therapeutic applications in companion animals.

Two bovine mammary epithelial cell lines, MAC-T and BME-UV1, were co-cultured either in Transwell® inserts or in 3D culture systems to comparatively investigate their potential to mimic the structure and function of a mammary alveolus. Using either Matrigel® or animal-friendly alternatives, i.e. rat collagen type I, a mixture of rat collagen type I and mouse laminin, Vitrogel® or matrix-free ultra-low attachment surfaces, both co-culture mammosphere development and tight junction formation were assessed. On Transwell® inserts, co-culture failed to establish a functional epithelial barrier. In contrast, varying patterns of cell–cell interaction and barrier organization emerged using the different 3D co-culture systems, providing new insights into the reconstruction of physiologically relevant and animal-friendly models of the bovine mammary gland.

CD94 is a natural killer (NK) cell receptor that also marks subsets of T cells, referred to as NKT cells. In humans, the role of CD94 as both an immune checkpoint and a potential therapeutic target has gained increasing attention. However, data about its expression in leukemia and lymphoma in dogs remain limited. This study aimed to explore CD94 expression in canine leukemia and nodal lymphoma, using a newly available anti-canine CD94 monoclonal antibody in a multicolor flow cytometry panel. Surplus blood and lymph node aspirate samples from eleven client-owned dogs (leukemia: n = 7, lymphoma n = 4) and two clinically healthy controls, were analyzed. The control dogs as well as most cases showed low CD94+ lymphocyte frequencies, consistent with a non-neoplastic population. However, markedly expanded CD94+ populations were identified in two out of four of the T cell chronic lymphocytic leukemia (T-CLL) cases. In one of them, the neoplastic population was uniformly CD3+CD8+CD94+, while the other showed a heterogeneous mixture of CD3+CD8+CD94+ and CD3−CD8+CD94+ lymphocytes. Our findings demonstrate that the canine-specific CD94 antibody can be applied to both blood and lymph node samples in a diagnostic flow cytometry setting. While CD94 expression was infrequent overall, its detection in a subset of T-CLL cases highlights the need for larger studies to determine its diagnostic and therapeutic value in canine leukemia and lymphoma.

ABSTRACT Background Combined measurement of functional, glomerular, and tubular markers in aging dogs is essential to detect early renal disease. Objectives Prospective longitudinal study to describe renal function and assess which biomarkers are associated with the development of early renal disease or death. Animals One hundred and twenty‐two apparently healthy senior and geriatric dogs. Methods Prospective longitudinal study. Renal function was evaluated at baseline (T0) and every 6‐12 months over 2 years, using systolic blood pressure measurements (SBP) and validated serum (creatinine, symmetric dimethylarginine, cystatin C [sCysC]), and urinary (specific gravity [USG], protein:creatinine [UPC], albumin:creatinine, retinol‐binding protein:creatinine [uRBPcr]) biomarkers. Glomerular filtration rate (GFR) was measured in a subgroup. Survival models were used to assess the predictive value of measured biomarkers at baseline for the onset of azotemic chronic kidney disease (CKD) or death, respectively. Results A total of 122 dogs were included; follow‐up was available in 106 (T12) and 92 (T24); and GFR was estimated in 18 (T0), 11 (T12), and 10 (T24) dogs. Throughout the study, 15/122 (12%) dogs showed evidence of non‐azotemic CKD, and in 11/106 (10%) dogs, azotemic CKD developed. Proteinuria was not associated with azotemic CKD, in contrast to muscle condition score, functional markers, and uRBPcr. Death was weakly associated with USG, UPC, and sCysC. Conclusions and Clinical Importance Over a 2‐year period, 20% (26/122) of older dogs developed CKD, mostly persistent renal proteinuria (15/122). Muscle wasting and functional markers combined with uRBPcr had the best predictive value for the onset of azotemic CKD in these older, previously apparently healthy dogs.

Neutrophil gelatinase-associated lipocalin (NGAL) and tissue inhibitor of metalloproteinase-2 (TIMP-2) have potential as early biomarkers for acute kidney injury (AKI) in dogs. Assess whether NGAL and TIMP-2 at admission (T0) and 24 h later (T1) identify survival in critically ill (CI) and AKI dogs, development of hospital-acquired AKI in CI dogs, and development of chronic kidney disease (CKD) in AKI dogs after 3 months. Sixty-two client-owned dogs: 10 healthy, 24 with AKI, and 28 CI. Prospective study with blood and urine samples collected at T0, T1, and up to 1 week in CI dogs, 1 month in healthy dogs, and 3 months in AKI dogs. Serum and urinary NGAL (sNGAL; uNGAL) and urinary TIMP-2 (uTIMP-2) were measured using validated ELISA kits. Dogs with AKI that did not survive had significantly higher uNGAL concentrations and u/sNGAL ratios at T0 compared with survivors (p = 0.05, n = 23; and p = 0.03, n = 21, respectively). In CI dogs, sNGAL was significantly higher in non-survivors at T0 and T1 compared with survivors (p = 0.02, n = 26; and p = 0.003, n = 26, respectively). At T0, normalized urinary tissue inhibitor of metalloproteinase-2 (unormTIMP-2) was significantly higher in non-survivor CI dogs compared with survivors (p = 0.04, n = 25). No significant differences were found for the other variables. In AKI dogs, uNGAL and u/sNGAL at T0, and in CI dogs, sNGAL at T0 and T1 and unormTIMP-2 at T0, were potential predictors of survival.

Designer cellulosomes (DCs) are precisely engineered multi-enzyme complexes aimed at lignocellulose saccharification. Achieving spontaneous designer cellulosome display on yeast cell surfaces has been a long-term objective to enhance consolidated bioprocesses with concurrent ethanol production. A "self-assembly" approach involves simultaneous scaffoldin display and docking enzyme secretion. However, challenges arise from the size and complexity of designer cellulosomes, coupled with the yeast cells' limited capacity for heterologous protein expression. A comprehensive examination of Saccharomyces cerevisiae as a host for DC expression remains unaddressed. We meticulously examined the capability of S. cerevisiae to produce and display a fluorescent protein complex, which mimics the designer cellulosome architecture and allows for convenient detection of all individual components on the cell surface, using flow cytometry and confocal microscopy. Population-wide analysis revealed a fluorescent protein complex production efficiency of approximately 10%. Single-cell analysis highlighted a clear mutual influence between the expression of scaffoldin and docking proteins, impacting cellular fitness. Newly emerging buds were identified as hotspots for scaffoldin display. The finite capacity of yeast cells to produce heterologous proteins was identified as a major bottleneck. While distributing the cellular load among multiple hosts within a synthetic yeast consortium can alleviate this burden, the use of fluorescent protein complex surface display has visualized the heterogeneity and constraints of S. cerevisiae as a host for designer cellulosome expression at the population and single cell level. This study provides a realistic assessment of the challenges in achieving efficient S. cerevisiae-based DC display for consolidated bioprocessing.IMPORTANCEEfficient and economically viable biomass conversion into fermentable sugars is a pivotal challenge in transitioning from a petroleum-based economy to a bio-economy. Drawing inspiration from nature, cellulosomes represent an exemplary solution for the effective digestion of lignocellulose. These multi-enzyme complexes can be precisely engineered to tailor their properties and transferred to the surface of yeast cells, which can subsequently ferment the sugars into bulk or fine chemicals. Achieving this transfer successfully necessitates a comprehensive understanding of how yeast cells can recombinantly produce and attach such multi-component complexes to their surface. This study employs a fluorescent surrogate to provide novel insights into the capabilities of yeast cells at both the single-cell and population levels.

The mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME), produced by Alternaria spp . , are common contaminants of food and feed and are a potential threat to animal and human health. To date, the most prominent data gaps for their comprehensive risk assessment concern information on their in vivo absorption, distribution, metabolism and excretion (ADME) and toxicokinetic behavior. The aim of this study was to determine the absolute oral bioavailability, quantitative toxicokinetic characteristics and biotransformation of AOH and AME in vivo in pigs, using crossover trials with intravenous and oral administration of a single dose of both mycotoxins at 2 mg/kg b.w. Plasma profiles of the mycotoxins and phase I and II metabolites were studied in the vena jugularis using UPLC-MS/MS and LC-HRMS methods. Furthermore, plasma from the vena portae was analyzed to study presystemic biotransformation. Urine was collected to determine the urinary excretion and metabolite profiles. Results reveal a low absolute oral bioavailability of AOH (15%) and AME (9%), caused by a low absorption and/or extensive first-pass biotransformation in the liver to mainly phase II, and to a lesser extent phase I metabolites. Quantitative toxicokinetic modeling of the IV data showed a high total body clearance for both AOH and AME (12.9 and 16.8 L/(h*kg b.w.), respectively), a high volume of distribution (4.97 and 5.15 L/kg b.w., respectively) and a short elimination half-life of 0.16 and 0.21 h, respectively. These findings may contribute to the risk assessment of AOH and AME and to the development of candidate biomarkers of exposure in biomonitoring studies since pigs are considered a suitable animal model to extrapolate to humans.

Liver-type fatty acid-binding protein (L-FABP) is expressed by several tissues, plays a role in fatty acid metabolism, and has antioxidant effects. Its renal expression is upregulated by stress. Urinary L-FABP (uL-FABP) is a promising kidney biomarker in people for detection of early acute and chronic kidney disease (CKD) and as a marker for progression in patients with glomerulonephritis. However, data on canine uL-FABP are currently limited. This prospective study was designed to examine canine tissue expression of L-FABP and to validate an ELISA to quantify uL-FABP in older dogs with or without early signs of CKD. Tissues of 4 recently euthanized dogs and 117 urine samples of 73 client-owned older dogs undergoing health screening were evaluated in the study. Immunohistochemistry was performed on kidney and liver tissues. Analytical validation of a commercially available ELISA for measurement of L-FABP in canine urine was performed (limit of detection, imprecision, specificity). The ELISA was used to measure L-FABP in stored urine samples from a cohort of older dogs. Dogs were found to express L-FABP, mostly in proximal tubular epithelial cells and in the periportal hepatocytes of the liver. Assay validation revealed poor sensitivity and imprecision for measurement of canine uL-FABP. Of the 117 urine samples analyzed, 98 were below the limit of detection (LOD; 7.30 ng/mL) and a further 5 were below the limit of quantification (LOQ; 16.20 ng/mL). The proximal tubules of dog kidneys express L-FABP, but the value of uL-FABP as tubular marker in older dogs warrants further study.

Vaccination against Mycoplasma hyopneumoniae is still carried out worldwide, but unfortunately current commercial vaccines only provide partial protection. Therefore, two M. hyopneumoniae strains were genetically modified by transposon-mediated gene disruption of mmsA and mnuA, encoding methylmalonate semialdehyde dehydrogenase and membrane nuclease A, respectively. We investigated how immune responses elicited by these genetically modified M. hyopneumoniae strains protected pigs against challenge infection. An endotracheal single dose vaccination with genetically modified M. hyopneumoniae strain 1 (ΔmmsA) or 2 (ΔmnuA), or physiological saline solution (Control) was followed by challenge infection. Piglets from ΔmnuA had a higher respiratory disease score post-vaccination, but this group coughed significantly less after challenge. Significantly fewer DNA copies of the challenge strains were observed in broncho-alveolar lavage fluid (BAL) from ΔmnuA after challenge. Two weeks post-challenge, significantly more BAL IgG and BAL IgA was observed in ΔmnuA, but at euthanasia significantly more IgA and less pro-inflammatory cytokines were detected in BAL from both vaccinated groups. Furthermore, a significantly lower percentage of IFN-γ+ and TNF-α+IFN-γ+ CD8+ T cells was observed after administration of ΔmnuA. The percentage of IFN-γ+ CD8+ T cells was significantly lower in ΔmmsA at euthanasia. To conclude, the results of this exploratory study show that a single endotracheal administration of ΔmnuA resulted in coughing post-vaccination, but reduced clinical signs post-challenge and challenge strain DNA load in BAL. Therefore, a strain mutated in the mnuA gene might be an interesting mutant strain that could be promising as a potential live vaccine candidate strain as it can reduce M. hyopneumoniae infection burden under field conditions.

Organoids have already shown great promise as research tools in human medicine. However, in veterinary medicine, such applications are limited and largely confined to canine organoids. In the Cross Health context, the potential of canine organoids lies in the translation to human diseases, such as cancer. This review provides a state-of-the-art, highlights the current challenges, and at first compares the reported culture conditions of canine organoids derived from both non-neoplastic and neoplastic tissue (i.e., tumoroids), identifying substantial gaps and discrepancies in used culture methods. We make a plea for the standardization of canine organoid culture characteristics and increased rigor in parameter reporting, which will ultimately enhance the reproducibility and applicability of canine organoids in both veterinary and human medicine, especially in the oncology field.

Triple-negative breast cancer (TNBC) patients exhibit variable responses to programmed death (PD)-ligand (L)1 blockade, largely determined by the ‘hot’ vs ‘cold’ state of the tumor immune microenvironment (TIME). We here characterized nine mouse TNBC models, relying on intraductal mammary gland inoculation of established mouse TNBC cell lines, with a heterogeneous TIME to study anti-PD-L1 resistance mechanisms. Complementary in vitro and in vivo screening classified the 4T1-hot-based model, a highly inflamed control through its immunogenic luciferase tag expression compared to the untagged 4T1-cold-based model, as displaying the ‘hottest’ TIME. However, both 4T1-based counterparts did not respond to anti-PD-L1, which was attributed to their immunosuppressive myeloid cell content, as well as upregulation of cancer-associated fibroblasts in the 4T1-hot and high PD-L1-expressing CXCL10+ tumor-associated macrophages in 4T1-cold primary tumors. These anti-PD-L1 adaptation mechanisms across TIME states, as captured by the mouse TNBC models, highlight specific cellular targets for future studies.

Critically ill patients admitted to the intensive care unit (ICU) frequently suffer from sepsis and severe multiple organ dysfunction with underlying widespread cell death. Pyroptosis and ferroptosis are regulated cell death forms that may serve as potential therapeutic targets. Pyroptosis is a major detrimental factor driving sepsis, which typically results in excessive oxidative stress potentially inducing ferroptotic organ injury. Here, we show that ICU patients with simultaneous pyro- and ferroptosis-positive signatures have the lowest survival probability. This is reflected by significantly elevated levels of pyroptosis-related biomarkers interleukin-1 receptor antagonist (IL-1Ra), IL-18, and growth and differentiation factor-15 (GDF15), as well as the ferroptosis-related biomarkers malondialdehyde (MDA) and catalytic iron (Fec). Moreover, combining these biomarkers with IL-1α, IL-6, IL-10, TNF, and chitinase-3-like protein 1 further improves clinical outcome prediction. The daily monitoring of pyro- and ferroptosis signatures reveals potential intervention opportunities, such as anakinra, tadekinig alfa, lead ferroptosis inhibitors, or a combination thereof. In summary, our findings demonstrate that a targeted biomarker panel enables predictive enrichment of ICU patients, paving the way for timely intervention strategies against pyroptosis or ferroptosis.

Macrophages play key roles in tissue homeostasis and regeneration-associated inflammation. Unlike humans, a reliable protocol to obtain and polarise equine monocyte-derived macrophages is lacking. In this study the polarisation of equine macrophages, derived from CD172a+ peripheral blood monocytes is described. After differentiation, IFN-γ/LPS or IL-4 were used to induce pro- and anti-inflammatory phenotypes, respectively. Evaluation criteria included morphology, mRNA (RT-qPCR) and protein expression (flow cytometry, immunofluorescence), nitric oxide and arginase production, cytokine secretion (multiplex), and functional effects of conditioned medium (CM). IFN-γ/LPS-stimulated cells exhibited a rounded morphology with cytoplasmic extensions, while IL-4 stimulation induced spindle-shaped and multinucleated giant cells. IFN-γ/LPS upregulated CXCL8, CD86, IL10 and TGFB1 mRNA, whereas IL-4 upregulated CXCL8, MRC1 and TGFB1. Polarisation was confirmed with IFN-γ/LPS-stimulated macrophages expressing CD86 and secreting TNFα and IL-1β, while IL-4-stimulation increased CD206 positivity and VEGFα expression. Increased proliferation and altered mRNA expression in tendon cells treated with 50% CM further validate the functional impact of macrophage polarisation. In summary, a robust protocol to obtain equine macrophages was developed, followed by in-depth characterization of their pro- and anti-inflammatory polarisation. Given the horse’s increasing relevance as large animal model, this research holds both a strong species-specific and translational value.

BACKGROUND:It is unknown if tumors or concomitant renal disease influence neutrophil gelatinase-associated lipocalin (NGAL) and symmetric dimethylarginine (SDMA) concentrations in tumor-bearing dogs. OBJECTIVES:Determine the effect of tumor presence, tumor type, and metastasis on concentrations of serum NGAL (sNGAL), SDMA, urinary NGAL (uNGAL), and uNGAL-to-creatinine ratio (uNGAL/Cr) in dogs with carcinoma or sarcoma without clinically relevant renal disease. ANIMALS:Twenty-one dogs with carcinoma, 18 with sarcoma, and 20 healthy age-controlled dogs. METHODS:Concentrations of sNGAL, SDMA, and uNGAL, and uNGAL/Cr ratio were measured from banked samples collected during a previous prospective study. Patient clinicopathological and histopathology records were reviewed, and those with renal azotemia or moderate to severe histopathological renal abnormalities were classified as having clinically relevant renal disease. Biomarker concentrations were compared between tumor-bearing dogs without clinically relevant renal disease and healthy age-controlled dogs. Additionally, comparisons were made between dogs with carcinoma and sarcoma, as well as between dogs with and without metastasis. Correlations between uNGAL and sNGAL concentrations, along with acute phase protein (APP) concentrations, were also analyzed. RESULTS:Tumor-bearing dogs without clinically relevant renal disease had increased uNGAL/Cr (p < 0.001), but not sNGAL, compared with healthy controls. Although median SDMA concentrations did not significantly differ between groups, increased concentrations were found in 32% of dogs with carcinoma and 20% of dogs with sarcoma. No differences were found between dogs with carcinoma and those with sarcoma, or between dogs with metastasis and those without. Urinary and serum NGAL concentrations were moderately correlated, while weak to no correlations were observed with APPs. CONCLUSION:Carcinomas and sarcomas, but not metastasis, influence uNGAL/Cr and SDMA concentrations in dogs.

BACKGROUND:Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS:Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS:Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS:Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

AbstractBackgroundCell cycle arrest biomarkers (tissue inhibitor of metalloproteinase‐2 [uTIMP‐2] and insulin‐like growth factor binding protein 7 [uIGFBP7]), and neutrophil gelatinase‐associated lipocalin (NGAL) variables are valuable biomarkers for early diagnosis of acute kidney injury (AKI) in people.ObjectivesTo evaluate uTIMP‐2, uIGFBP7, fractional excretion of NGAL (FeNGAL), and urinary to serum NGAL ratio (u/sNGAL) in healthy dogs, dogs with AKI, dogs with chronic kidney disease (CKD), and critically ill (CI) dogs.AnimalsForty‐two client‐owned dogs (healthy, n = 10; AKI, n = 11; CKD, n = 11; CI, n = 10).MethodsProspective, observational study. After assessment of routine renal biomarkers, stress (uTIMP‐2, uIGFBP7) and damage (NGAL) biomarkers were measured, using ELISA kits, and normalized to urinary creatinine (uCr).ResultsNormalized uTIMP‐2 and [uTIMP‐2] × [uIGFBP7]/uCr were significantly higher in the AKI group (median 151.9 [range, 2.2‐534.2] and 62.9 [1.1‐266.8] pg/mL respectively), compared to healthy dogs (0.3 [0.2‐74.7]; P < .001 and 0.16 [0.1‐58.1] pg/mL; P < .001), dogs with CKD (0.7 [0.3‐742.5]; P = .04 and 0.37 [0.2‐180.1] pg/mL; P = .03) and CI dogs (1.9 [0.2‐37.0]; P = .03 and 0.8 [0.1‐16.1] pg/mL; P = .02). Fractional excretion of NGAL was significantly higher in dogs with AKI (54.17 [7.93‐155.32] %), than in healthy (0.03 [0.01‐0.21] %; P < .001) and CI dogs (3.05 [0.05‐28.86] %; P = .02).Conclusions and Clinical ImportanceNormalized uTIMP‐2, [uTIMP‐2] × [uIGFBP7]/uCr, and FeNGAL can be valuable renal biomarkers for early diagnosis of AKI in dogs.

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 μg) combined with either a low (23.5 μg) or high (235.0 μg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog10; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. KEY POINTS: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.

Bacteriophage-derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic-resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high-throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram-positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time-kill assays in phosphate-buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta-lactam penicillin used to intramammarily treat bovine Gram-positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add-on in the treatment of bovine streptococcal mastitis.

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Acute kidney injury (AKI) is common after pediatric cardiac surgery (CS). Several urine biomarkers have been validated to detect AKI earlier. The objective of this study was to evaluate urine CHI3L1, NGAL, TIMP-2, IGFBP7, and NephroCheck® as predictors for AKI ≥ 1 in pediatric CS after 48 h and AKI ≥ 2 after 12 h. Pediatric patients (age < 18 year; body weight ≥ 2 kg) requiring CS were prospectively included. Urine CHI3L1, NGAL, TIMP-2, IGFBP7, and NephroCheck® were measured during surgery and intensive care unit (ICU) stay and corrected for urine dilution. One hundred and one pediatric patients were included. AKI ≥ 1 within 48 h after ICU admission occurred in 62.4% and AKI ≥ 2 within 12 h in 30.7%. All damage biomarkers predicted AKI ≥ 1 within 48 h after ICU admission, when corrected for urine dilution: CHI3L1 (AUC-ROC: 0.642 (95% CI, 0.535–0.741)), NGAL (0.765 (0.664–0.848)), TIMP-2 (0.778 (0.662–0.868)), IGFBP7 (0.796 (0.682–0.883)), NephroCheck® (0.734 (0.614–0.832)). Similarly, AKI ≥ 2 within 12 h was predicted by all damage biomarkers when corrected for urine dilution: uCHI3L1 (AUC-ROC: 0.686 (95% CI, 0.580–0.780)), NGAL (0.714 (0.609–0.804)), TIMP-2 (0.830 (0.722–0.909)), IGFBP7 (0.834 (0.725–0.912)), NephroCheck® (0.774 (0.658–0.865)). After pediatric cardiac surgery, the damage biomarkers urine CHI3L1, NGAL, TIMP-2, IGFBP7, and NephroCheck® reliably predict AKI after correction for urine dilution.