All antibodies approved for cancer therapy are monoclonal IgGs but the biology of IgE, supported by comparative preclinical data, offers the potential for enhanced effector cell potency. Here we report a Phase I dose escalation trial (NCT02546921) with the primary objective of exploring the safety and tolerability of MOv18 IgE, a chimeric first-in-class IgE antibody, in patients with tumours expressing the relevant antigen, folate receptor-alpha. The trial incorporated skin prick and basophil activation tests (BAT) to select patients at lowest risk of allergic toxicity. Secondary objectives were exploration of anti-tumour activity, recommended Phase II dose, and pharmacokinetics. Dose escalation ranged from 70 μg–12 mg. The most common toxicity of MOv18 IgE is transient urticaria. A single patient experienced anaphylaxis, likely explained by detection of circulating basophils at baseline that could be activated by MOv18 IgE. The BAT assay was used to avoid enrolling further patients with reactive basophils. The safety profile is tolerable and maximum tolerated dose has not been reached, with evidence of anti-tumour activity observed in a patient with ovarian cancer. These results demonstrate the potential of IgE therapy for cancer. IgE antibodies have shown anti-tumor activity, even superior to IgG, in preclinical models. However, all monoclonal antibodies in clinical use for cancer therapy are members of the IgG class. Here the authors report the results of a phase I clinical trial of a chimeric monoclonal IgE antibody, specific for the folate receptor-alpha, in patients with advanced solid cancer. Circulating levels of IgE are much lower than IgG, and IgE has evolved to provide immune surveillance in tissues4. The mechanism of action of therapeutic antibodies is mediated in part by their engagement with cognate Fc receptors on specific immune effector cell populations7. The structure of the Fc domain of IgE differs from that of IgG, and it binds with very high affinity to FcɛRI receptors, expressed on cells including monocytes/macrophages, mast cells, basophils, and dendritic cells, resulting in long retention of antibody by these effector cells in the absence of immune complex formation8  was chosen as the target for clinical testing of IgE therapy because of its presence on the cell membrane Fig.  of a ran25. Secondly, pre-dose basophil activation tests (BAT) were performed as previously described26 on fresh whole blood from each patient using MOv18 IgE. This test was designed to model ex vivo the ability of MOv18 IgE to activate circulating cells, with the expectation that the BAT would function as an assay predictive of anaphylaxis. The safety profile of MOv18 IgE is tolerable, the most common toxicities being cutaneous, and there is evidence of anti-tumour activity.We have designed and conducted a Phase I trial of MOv18 IgE, a chimeric monoclonal IgE antibody specific for FRα, in patients with tumours expressing this antigen. Procedures prior to intravenous administration of MOv18 IgE were included to mitigate the possibility of unwanted IgE-mediated effects. First, skin prick testing with a solution of MOv18 IgE antibody was undertaken, with a rationale of detecting putative soluble factors, possibly originating from the target tumour, that might cross-link the antibody once bound to Fc receptors. This could result in mast cell and basophil degranulation and possible anaphylaxis. If present these factors would be expected to produce a wheal and flare reaction on skin prick testing, analogous to those seen in classical cutaneous allergy testing performed with solutions of antigensn = 21), tubal carcinoma (n = 3) or endometrial cancer (n = 2). A low prevalence of FRα expression was observed in other tumour types screened using our validated assay. 24 patients (median age 61) were treated with up to six weekly MOv18 IgE infusions  and 7(ii), respectively). No further DLTs occurred and a maximum tolerated dose has not been defined.Patients with any solid tumour expressing FRα using our immunohistochemical assay Fig.  were elions Fig.  after inons Fig. . PatientMOv18 IgE was generally well tolerated with the great majority of adverse events being low grade . The most common events were localised cutaneous toxicities including urticaria, pruritus and rash Fig. ; cutaneoOne patient treated in the 500 μg cohort experienced an episode of transient hypotension during their first infusion, a few minutes following the onset of urticaria. Treatment was discontinued and the hypotension responded immediately to adrenaline and the administration of intravenous hydrocortisone, antihistamine and saline. The diagnosis of grade 3 anaphylaxis was confirmed by the detection of elevated serum tryptase following this episode Fig. , which wSkin prick testing was routinely performed before each intravenous dose. In general, the reaction to MOv18 IgE in this test was indistinguishable from the negative diluent control, although a wheal reaction of up to 3 mm was also considered negative Fig. . A positFree FRα protein was detected at one or more time points  in the serum of eight patients, although in the remainder it was undetectable throughout . Three patients developed anti-alpha-GAL antibodies  but these were not associated with any clinical signs of anaphylaxis and were no longer measurable at the 28-day follow-up  and Cmax, was observed to increase in a dose-proportional manner  ex vivo on whole unfractionated blood from each patient just prior to dosing, with a rationale of addressing concerns that MOv18 IgE bound to endogenous Fcε receptors might be cross-linked by endogenous antibodies or other circulating factors. The BAT results remained negative in all patients with the exception of one who developed anaphylaxis on their first exposure to MOv18 IgE. The episode resolved rapidly and did not recur after withdrawal of drug. This patient demonstrated a positive BAT at baseline, prior to any exposure to MOv18 IgE. We hypothesise that, in this single patient, both anaphylaxis and the positive BAT at baseline reflected the presence of an endogenous circulating factor able to crosslink the MOv18 IgE antibodies bound to receptors on the surface of effector cells. One such candidate may be autoantibodies to the oligosaccharide galactose-α−1,3-galactose : subjects with an endogenous IgE response against this post-translational modification on a therapeutic antibody have been reported to develop Type 1 hypersensitivity reactions during treatment with some monoclonal antibodies26. The prevalence of positive baseline BAT therefore appears low in this population of patients with cancer, but screening for baseline BAT positivity may be useful in future to exclude these infrequent patients from IgE therapy and the associated risk of anaphylaxis.Following this single case of anaphylaxis, the trial protocol was amended to exclude any further patients found to have a positive baseline BAT, and no further anaphylaxis was observed. In a separate observational study of blood samples drawn from a series of patients with ovarian cancer, we observed a positive BAT using MOv18 IgE in only one of 42 individuals38, was observed in any of the remaining patients who experienced urticaria. Cutaneous reactions were not seen in animal models of IgE therapy39, and the mechanism accounting for the common occurrence of urticaria, in the absence of anaphylaxis, in humans receiving MOv18 IgE is currently unclear. Our own data, consistent with the literature41, suggest that FRα is not expressed in skin , and although distribution to the skin of healthy subjects was claimed in a clinical study of a fluorescently labelled anti-FRα agent, this was attributed to drug aggregation42. MOv18 IgE does not bind to human skin ex vivo, and histological examination of biopsied urticarial skin from a patient in this trial did not demonstrate prominent infiltration by basophils .The most common adverse events associated with MOv18 IgE were cutaneous, with some patients experiencing a diffuse patchy macular erythematous rash, with or without pruritus, but with urticaria the most frequent toxicity. Although the extent of this rash varied greatly between patients, it was tolerable and transient. Urticaria usually recurred with subsequent doses, but in general it was more prominent with first dose administration and became less obvious with subsequent doses. It is possible this could have been a result of the use of secondary prophylaxis with steroid and antihistamine. Urticaria was associated with symptoms or signs of anaphylaxis only in the single patient referred to above with a positive BAT at baseline. No elevation of the serum tryptase concentration, a diagnostic requirement in cases of anaphylaxis43, but is expressed in a range of solid tumours18. Several IgG-based antibody therapies targeted to FRα have been tested clinically44, with the recent approval of mirvetuximab soravtansine further validating this target24. Our pre-clinical evidence supporting superior anti-tumour activity of FRα-targeting IgE suggests a route to improvement of earlier clinical results with anti-FRα IgG. Tumour types most commonly expressing FRα with the IHC used in this trial were epithelial ovarian/tubal and endometrial cancers. We observed preliminary evidence of MOv18 IgE anti-tumour activity in high grade serous ovarian carcinoma, with a fall in CA125 and tumour shrinkage in a patient with chemotherapy-resistant disease. If this is confirmed in subsequent clinical development, this antibody may join the growing immunotherapy armamentarium in FRα-positive cancers45. More broadly, these results offer the opportunity to generate a new class of IgE antibody cancer therapies specific for other antigens already validated as targets for IgG drugs.FRα was chosen as the target for testing IgE therapy in a clinical trial. This tumour-associated antigen is present at very low levels in normal tissuesThis was an open-label, dose-escalating Phase I study of MOv18 IgE conducted at 4 sites in the United Kingdom. The study was undertaken under the sponsorship and management of the Cancer Research UK Centre for Drug Development, conducted in accordance with International Council for Harmonisation Good Clinical Practice guidelines and ethical principles of the Declaration of Helsinki, and approved by the UK Medicines and Healthcare products Regulatory Agency and National Health Service Health Research Authority . Participants provided written informed consent prior to the conduct of any study procedures. Antibody was administered as an intravenous infusion weekly for a six-week treatment period. In the absence of disease progression or intolerable treatment-related toxicity, patients were offered the option to continue two-weekly dosing for a further maintenance period.Screening tests at baseline included physical examination, computerised tomography (CT), standard tests of organ function, beta-tryptase, endogenous IgE level, tumour FRα expression, basophil activation test, and intradermal or skin-prick test. Safety assessments were repeated prior to each further dose throughout the study. All patients receiving at least 1 intravenous dose of MOv18 IgE were evaluable for safety. Anti-tumour activity was assessed using standard tumour markers where relevant , and CT according to RECIST version 1.1 at intervals of 6 weeks. Laboratory endpoints were analysed using SSPS version 29 and presented with Graphpad Prism version 9.5.46. Briefly, sections of formalin-fixed, paraffin embedded tissue were pre-treated using citrate-based heat-induced antigen retrieval (CC2) for 92 min, followed by staining with FRα antibody  at a dilution of 1:500 using the Ventana Benchmark Ultra (Roche) automatic staining system. Positive (FRα expressing ovarian tumour tissue) and negative  controls were included. Of the 445 individuals assessed for tumour expression of FRα, 37% were deemed to be positive. Other inclusion criteria were: measurable or evaluable disease; resolution of any residual toxic effects, aside from sensory neuropathy, related to prior anti-cancer therapy to grade 1 or lower (as defined by the Common Terminology Criteria for Adverse Events (CTCAE), version 4.0); no chemotherapy, radiotherapy, biological therapy, or hormone for at least four weeks, or five half-lives, prior to receiving the study treatment; an Eastern Cooperative Oncology Group (ECOG) performance score of 0 or 1; adequate bone marrow, liver, and kidney function. Patients with treated and stable brain metastases were allowed. Exclusion criteria included: a history of congenital or acquired immunosuppression, including an ongoing requirement for systemic steroid therapy; high risk or uncontrolled asthma; a history of anaphylaxis; elevated serum tryptase at baseline; a history of laryngeal oedema, known HIV, hepatitis B or C infection; pregnancy or breast-feeding; elevated risk in the event of anaphylaxis because of heart failure, uncontrolled infection, vascular disease, previous cerebrovascular accident, extensive lung metastases or pleural effusion; concurrent medication with drugs likely to inhibit or augment the effects of adrenaline, including beta-blockers, tricyclic antidepressants and monoamine oxidase inhibitors.Recruitment and treatment took place between 23 February 2016 and 20 April 2021. Eligible patients had advanced or metastatic solid tumours not suitable for alternative standard treatment and were over 16 years of age. Immunohistochemical evidence of tumour FRα membrane expression was required (based on a criterion of ≥5% tumour cells with membrane positivity)Drug was supplied (Cancer Research UK) and manufactured as single-use aseptic 1 mL fill in 2 mL glass vials containing a solution of MOv18 IgE at a concentration of 1 mg/mL at pH6.5 with 0.1 M sodium citrate, 30 g/L L-arginine, 50 g/L sucrose and 0.02% polysorbate 20 in water for injection. Stock was stored at 5 ± 3 °C, initially diluted to a concentration of 100 μg/mL in 0.9% sodium chloride, then infused over 90 min through a peripheral line in a total volume of 250 ml 0.9% saline. A shorter infusion time for subsequent doses of 60 min was allowed if no adverse reactions are encountered. Vital signs were monitored during the infusion and at regular intervals afterwards. Patients were hospitalised overnight following the first administration to facilitate safety monitoring procedures.The protocol-defined treatment regimen was 6 doses of MOv18 IgE administered weekly, following which disease response was reassessed using CT. Patients tolerating treatment and appearing to benefit from treatment were offered the option of continuing into a maintenance phase of two-weekly dosing for a further 6 weeks Fig. .47. An additional 2–5 patients could be added to each cohort in the event of emergent toxicity. Thereafter, three patients were enrolled per cohort, with an additional three patients added to a cohort if needed for toxicity. The starting dose of 70 μg was selected to be lower than endogenous IgE, and was based on the relatively low physiological IgE serum levels and high affinity for receptors on effector cells. It also reflected partial occupancy of Fc receptors by endogenous IgE, meaning that anti-tumour IgE could engage and activate immune cells at concentrations much lower than the equivalent IgG. Each patient was assessed for safety and dose-limiting toxicities (DLTs). DLT was defined as any grade 3/4 non-haematological adverse event  occurring in the first 3 weeks of MOv18 IgE administration. Haematological DLTs were defined as grade 4 neutropenia or thrombocytopenia that was prolonged, or grade 3/4 neutropenia associated with fever or infection.The planned escalation in successive flat dosing cohorts was 70 μg, 250 μg, 500 μg, 700 μg, 1.5 mg, 3 mg, 6 mg and 12 mg total protein. An accelerated dose escalation scheme, starting with single-patient cohorts, was planned up to cohort 5 (1.5 mg)https://www.bsaci.org/Guidelines/SOPs], but subsequently SPT was preferred due to serial positive results with intradermal testing, which did not appear to discriminate those at risk.Skin prick testing (SPT) with a solution of MOv18 IgE antibody was undertaken in patients prior to each intravenous administration. Positive histamine and negative saline controls were included, and the presence of a wheal reaction in response to the positive control was required for a test to be considered valid Fig. . Concomi26 on fresh whole blood using aliquots of the trial supply of MOv18 IgE, and a laboratory standard solution. Briefly, basophils in unfractionated whole blood samples were incubated with positive controls anti-FcεRI, fMLP (each as part of Buhlmann FlowCAST kit FK-CCR-U), and anti-IgE (Dako Aligent A0094), or MOv18 and control IgE antibodies (non-FRα-reactive IgE antibodies) for 30 min at 37 °C. Staining antibodies (Buhlmann FlowCAST kit FK-CCR-U) were used for CCR3 (to identify basophils) and CD63 (as a marker of activation)48. Flow gating strategy is shown in Supplementary Fig. Similarly, basophil activation tests (BAT) were performed as previously described38. The protocol mandated tryptase measurement at baseline, at the end of each infusion, and following any infusion-related event, primarily to distinguish cytokine release from anaphylaxis. It was also measured following the onset of urticaria, which can occur as a feature of anaphylaxis49.No features of anaphylaxis or other manifestation of allergic toxicity were observed in preclinical animal models, but in addition to the risk mitigation steps described above, serial measurements of serum tryptase were included in this trial. Tryptase is released by degranulation of activated effector cells, and elevation is characteristic of anaphylaxis17 and to the proposed mechanism of anti-tumour activity of MOv18 IgE50. Where possible, serial tumour biopsies were collected for analysis of changes in the tumour microenvironment.Patient specimens were also collected for pharmacodynamic analysis. Serum samples were collected pre- and post-dose for multiplex analysis of a cytokine panel (Randox) relevant to IgE biologyAnti-drug antibody (ADA) samples were drawn at baseline and 2 and 5 weeks following initiation of weekly dosing. Additional samples were collected 28 days after the last dose was administered and following any suspected infusion reaction. Serum samples were stored frozen at −80 °C prior to analysis using a bridging ELISA assay with MOV18 IgE to capture, and biotinylated MOv18 IgE followed by streptavidin-HRP (BD 554066) to detect the presence of ADAs. A confirmatory assay was performed on all positive samples, pre-incubating with drug to distinguish specific responses from false positives.37. Briefly, FRα in patient sera was captured by mouse anti-human FRα IgG1 (R&D Systems MAB5646), and detected by biotinylated polyclonal goat anti-human FRα antibody (R&D Systems BAF5646) followed by streptavidin-HRP (Pierce 21130). Similarly, anti-FRα antibodies were captured by recombinant FRα (BioTechne 5646-FR) and detected by anti-IgG1-HRP antibody (Jackson Immuno Research 109-036-098). Levels were quantified by recombinant FRα (BioTechne 5646-FR) and anti-FRα IgG1 (in house) standard curves, with lower levels of quantification (LLOQ) of 6.25 ng/ml and 3.125 ng/ml in serum, respectively.Further blood samples were drawn at baseline, 3 weeks following initiation of weekly dosing and 28 days after the last dose, as well as following any observed adverse events. Serum was separated and stored at −80°C prior to evaluation of circulating soluble FRα and anti-FRα antibodies by ELISA, as previously describedt½), maximum concentration (Cmax), time of peak serum concentration (Tmax), and area under the serum concentration-time curve (AUC).Pharmacokinetic blood samples were drawn immediately pre-dose and at 0.5, 2, 4, 6, and 24 h, then 7 days, following the first dose, and 28 and 70 days after the final dose. Serum was separated from blood and stored at −70°C prior to analysis for MOv18 IgE concentrations by indirect ELISA, using a fully validated assay. Briefly, MOv18 IgE antibodies were captured by recombinant FRα (BioTechne 5646-FR) and detected by an anti-IgE-HRP detection antibody (Sigma A9667). Levels were quantified by a MOv18 IgE standard curve with a limit of quantification of 2.5 ng/ml and a dynamic range of 2.5–200 ng/mL in serum. The following pharmacokinetic parameters were evaluated: terminal half-life calculated from the terminal slope of the log concentration–time curve (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Desulfobacterota , Myxococcota, and Sva0485, whereas Gammaproteobacteria, Alphaproteobacteria, Bacteroidota, and NB1-j prevail at the heavy ice cover station. In the ferruginous zone, Sva1033 was the dominant member of Desulfuromonadales for all stations and, along with eleven other taxa, showed significant positive correlations with dissolved Fe concentrations, suggesting a significant role in iron reduction or an ecological relationship with iron reducers. Our results indicate that sea ice cover and its effect on organic carbon fluxes are the major drivers for changes in benthic microbial communities, favoring potential iron reducers at stations with increased organic matter fluxes.The area around the Antarctic Peninsula (AP) is facing rapid climatic and environmental changes, with so far unknown impacts on the benthic microbial communities of the continental shelves. In this study, we investigated the impact of contrasting sea ice cover on microbial community compositions in surface sediments from five stations along the eastern shelf of the AP using 16S ribosomal RNA (rRNA) gene sequencing. Redox conditions in sediments with long ice-free periods are characterized by a prevailing ferruginous zone, whereas a comparatively broad upper oxic zone is present at the heavily ice-covered station. Low ice cover stations were highly dominated by microbial communities of  In, e.g., ,9,10,11.n, e.g., ,13,14,15n, e.g., ,11,16. Tn, e.g., ,16,17,18n, e.g., ,20. Thern, e.g., ,26,27,28In the area of the Antarctic Peninsula (AP), the amount of organic matter originating from surface-water primary production is highly variable—both temporally and spatially—and depends mainly on the availability of light, which is regulated by season and sea ice cover ,30,31. D2 and enhanced accumulation of phytodetritus on the sea floor, a process known as the biological carbon pump [−1 of bioavailable Fe [−1) [−1) [−1) [Another factor regulating the production and subsequent deposition of OM over large parts of the Southern Ocean is the availability of dissolved iron (DFe), a limiting micronutrient for phytoplankton productivity, e.g., ,38. Ironbon pump ,40. Ironlable Fe , followe−1) [−1) ,42, and −1) [−1) . The imp−1) [−1) ,44,45, a−1) [−1) ,47,48. T−1) [−1) ,49.Desulfuromonadales  [Sva1033, a clade of Desulfuromonadales, was identified by Ravenschlag et al. [Sva1033 has iron-reducing capabilities.In shelf sediments, dissimilatory microbial iron reduction (DIR) plays a key role in the remineralization of organic matter ,45,50,51obacter) ,55,56,57g et al.  and Wundg et al.  using RN2S concentrations marked the beginning of the sulfidic zone [Sedimentary redox conditions along the eastern shelf of the AP have recently been investigated by Baloza et al. , who foudic zone .The present work on the microbial communities is complementary to the geochemical findings of Baloza et al.  and is bDuring research cruise PS118 with the German research vessel RV POLARSTERN (February–April 2019), sediments were collected from five stations along a 400-nautical mile transect from the eastern Antarctic Peninsula to the West of the South Orkney Islands ; Table 1The DNA was extracted from 0.25 g of wet sediment using the DNeasy PowerSoil Kit  following the protocol provided by the manufacturer. Amplicon sequencing was conducted on an Illumina MiSeq machine at AWI laboratory. For the 16S rRNA gene amplicon library preparation, we used the bacterial primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (50-GACTACHVGGGTATC TAATCC-30), which amplify the 16S rRNA gene hypervariable region V3–V4 in Bacteria (400–425 bp fragment length). The amplicon library was sequenced with MiSeq v3 chemistry in a 2 × 300 bp paired-end run with >50,000 reads per library, following the standard instructions of the 16S Metagenomic Sequencing Library Preparation protocol .The quality cleaning of the sequences was performed using several software tools. The first tool was Cutadapt , which wp-values < 0.05) with ‘vegan’ v2.5.7 [p-value < 0.05).All statistical analyses were conducted using RStudio v4.0.2; R Core . Sample ’ v2.5.7 . Geochem’ v2.5.7 . The sigp < 0.05. Furthermore, the environment coverage for the most closely related sequences (>99% similarity) to ASV highly abundant in the ferruginous zone was identified with BLASTn  and reported for each ASV.In order to identify taxonomical associations with iron cycling of all shelf stations, the Metastats-test-based differential abundance analysis of taxa was performed using the ALDEx2 package v1.20.0  at a sigUsing Illumina 16S rRNA amplicon sequencing of the V3–V4 hypervariable region, we obtained a final dataset of 4,252,065 reads (amplicons) in 90 samples, which were assigned to 38,655 ASVs belonging to bacterial taxonomies of 64 phyla, 162 classes, 378 orders, 536 families, and 786 genera. Rarefaction profiles of 16S rRNA gene sequences reached a plateau for most of the samples . Sedimen−4). In contrast, the original dataset and the normalized dataset to the smallest number of reads did not significantly correlate . Based on this result, the original dataset was used for further analysis.Sample read counts ranged from 942 to 250,824 per sample , the medp < 0.001). This result suggests that sea ice cover is a variable affecting the microbial community composition in the AP shelf sediments. To further test the contribution of sea ice cover in explaining differences in the microbial community, a permanova test was carried out, also including sediment depth as an explanatory factor, which is an important environmental constraint for microbes (p < 0.0001) and can explain 4%  to 13% (when testing the surface layers only) of the variation between microbial communities. Sediment depth was also tested as another factor affecting the microbial community structure. The results showed a significant difference between sediment depths (p < 0.0001), explaining 9% of the variance.Non-metric multidimensional scaling of ASVs did not show any clear pattern between stations A. Howevemicrobes . The perp < 0.001, Df: 7, 81). The factors of O2, DFe, DIC, SO42−, Fe(III)/Al, S/Al, and the C/N ratio were included as explanatory variables in the model and together explained 20% of the total variation in the bacterial community. The variance inflation factor between these variables was below five (VIF = 4.3), indicating independence between variables. The total organic carbon content (TOC) was not used as an explanatory variable due to collinearity to other variables such as DIC, SO42−, Fe (III)/Al and S/Al. NH4+. H2S was also removed from this data analysis as it correlated with DIC and SO4 2−, respectively. However, we used the sulfur content in the solid phase (S/Al) instead of H2S as an indication of sulfate reduction and accumulation of pyrite.In order to identify the potential geochemical parameters that correlate with the microbial communities across all sites, a dbRDA was performed  and DFe concentrations , followed by S/Al , Fe/Al , then C/N ratio , DIC , and SO42− . In accordance with the NMDS ordination  , except for order_B2M28, where p = 0.08  were performed using BLASTn (NCBI website) . The mosThe Antarctic Peninsula is projected to undergo profound climatic and environmental changes affecting seasonal sea ice cover, water column stratification, terrestrial meltwater run-off, and related nutrient input, and thus the conditions for primary production, organic carbon (OC) export, and benthic remineralization ,82,83. T−2 d−1 was measured for the station with heavy ice cover, explained by limited light availability and thus low surface-water primary production. In contrast, the locations with low sea ice cover showed high carbon supply rates of up to 13.0 mmol C m−2 d−1, indicating that primary and export production are both enhanced when both light and water column stratification is sufficient to support phytoplankton growth. This scenario is supported by known large phytoplankton blooms and high primary production during spring/summer seasons on the marginal ice zone along the AP [In this study, we observed that benthic microbial communities are significantly different between sites with high and low ice cover. Recently, a biogeochemical study of sediments from the exact same stations  describeg the AP ,85,86,87g the AP ,34. Currg the AP  also foug the AP . Conversg the AP . In addi−2 d−1 and a deeper oxygen penetration depth compared to stations with low ice cover  (−2 d−1), resulting in a more condensed redox zonation and high concentrations of dissolved Fe (>400 μM at Shelf St4) close to the sediment surface  , resulti surface . Below tion zone . In accore water B, which re water B. This iter , which harbor many species of iron reducer taxa [Desulfobacterota (mostly Desulfobacteria and Desulfobulbia), Myxococcota (mostly Polyangia), and Sva0485 were abundant near the sediment surface of low ice cover (>20%) and at greater depth (14 cm depth) (>12%). Similarly, some of those taxa have been previously observed in shallower depths in more reducing shelf sediments of the Sub-Antarctic island of South Georgia [Microbial communities in surface sediments  of all stations are dominated by bacterial groups belonging to c matter ,75,88,89cer taxa ,26,54,90 Georgia . Unlike Furthermore, the high relative abundance of sulfate reducers in the ferruginous zone at the sites with low ice cover can be explained by the tight link between the biogeochemical cycles of iron and sulfur. For this reason, Fe liberation into the pore water can result either from dissimilatory iron reduction (DIR) or happen abiotically related to sulfide oxidation by Fe(III) reduction, e.g., . The tig−2 d−1), while a low upward flux of DFe was observed at the heavy ice cover station (17.7 µmol m−2 d−1). The steep concentration gradients of DFe close to the sediment surface indicate that more DFe might escape from shelf sediments into the water column highlighting the importance of sediments underlying low ice cover as a potential source for limiting nutrients to the shelf waters [The anoxic shelf sediments have recently been considered a potential source of bioavailable Fe to Antarctic coastal waters and beyond . Along tf waters . To idenSva1033, a clade of Desulfuromonadia, is tightly coupled to the increase in the dissolved iron concentration showing the steepest slope in the linear regression (Sva1033 decreased (<2%) near to sediment surface while it increased (>4%) below 5 cm depth in the ferruginous zone  are SEEP-SRB4 (class Desulfobulbia), Persicirhabdus (class Verrucomicrobiae), Halioglobus, order_B2M28 and OM60 (NOR5) clade (class Gammaproteobacteria), Maribacter and Lutimonas (class Bacteroidia) and class Polyangia  to microbial communities reveals the importance of sediments underlying low ice cover as a potential source of dissolved iron to shelf waters. In this regard, the findings reported here expand our knowledge about changes induced by the increase of OM load to microbial community composition in AP shelf sediments under contracting sea ice cover and stimulate future research to better elucidate the role of microbial iron reducers in the biogeochemical iron cycle.Our study reveals that sea ice cover and associated carbon burial fluxes explain up to 13% of the variability between microbial communities in the AP shelf sediments. At all stations,


New generation 3-dimensional matrix array intracardiac echocardiography catheters have become commercially available recently, increasing image plane options compared to 2-dimensional and older generation 3-dimensional intracardiac echocardiography catheters. They are beginning to play an important role in structural heart interventions, especially for transcatheter tricuspid interventions, due to advantages in some situations that increase image quality over transesophageal echocardiography. Transcatheter structural heart interventions rely heavily on live echocardiographic guidance performed by interventional imaging specialists to achieve safe, efficient and effective procedures . The mosNew generation 3-dimensional matrix array intracardiac echocardiography (ICE) catheters have become commercially available in the United States serving as an important adjunctive imaging modality to TEE in structural heart interventions. ICE has several advantages over TEE, specifically offering near field imaging and avoiding intracardiac acoustic shadowing in some situations. There are also reduced sedation requirements, potentially allowing certain procedures to be performed without general anesthesia and mechanical ventilation.Three 3-dimensional ICE platforms are already commercially available in the United States. All three technologies are capable of single plane 2-dimensional imaging, biplane imaging, live 3-dimensional imaging and live 3-dimensional multi-planar reconstruction.The Siemens AcuNav Volume  has been available for a longer period of time than the newer matrix array probes. The AcuNav Volume is a 12.5 French system with a 128-element helical transducer element array. It can provide a live 3-dimensional volume size up to 90 × 50 degrees. The Siemens ACUSON SC2000 echocardiography console is used to generate and control 2- and 3-dimensional images. Four degrees of flexion including anterior, posterior, left and right are possible.The Philips VeriSight Pro  catheter is a 9 French system with an 840-element matrix array. The maximum 3-dimensional volume size is 90 × 90 degrees. It is compatible with the Philips Epiq CVxi, CVx and 7C consoles. Anterior, posterior, left and right flexion are controlled using two rotary knobs on the probe handle.The Biosense Webster NuVision  catheter is a 10 French system with an 840-element matrix array. The maximum 3-dimensional volume size is also 90 × 90 degrees. It is compatible with the GE E95 and S70N  consoles. Uniquely, while there is only anterior and posterior flexion, the probe has the capability of rotating the transducer array 360 degrees around the long axis of the catheter independently of the catheter itself.It is important to recognize that 3-dimensional ICE is not identical to 3-dimensional TEE and important differences exist. 3-dimensional TEE probes have much larger matrix arrays , which provide better spatial and temporal resolution. The maximum 3-dimensional volume size is also larger on TEE. Biplane image quality is more consistent across all angles relative to the transducer array on TEE, vs. ICE which suffers spatial resolution degradation perpendicular to the long axis of the catheter (at 90 degrees on the omniplane angle) due to physical transducer array limitations. As a result, optimal biplane imaging on 3-dimensional ICE is achieved when the imaging planes are closer to diagonal across the matrix array, such as at 45 and 135 (or −45 and +45) degrees. Lastly, while TEE probes are multi-use, 3-dimensional ICE catheters are single use.With respect to the omniplane angle, 0 degrees on an ICE probe corresponds to the imaging plane that is parallel to the long axis of the catheter, whereas 0 degrees on a TEE probe corresponds to the imaging plane that is perpendicular to the long axis of the probe.One of the natural questions that is raised by the introduction of 3-dimensional ICE into the transcatheter intervention space is how the imaging expert fits into the workflow of this imaging modality. While some may argue that these catheters open the possibility of minimizing personnel and removing the interventional imager from the room, there is a considerable amount of expertise in image optimization, biplane and multi-planar reconstruction generation, image interpretation, and procedural decision making that an expert imager can contribute. Additionally, fusion imaging systems are expected to incorporate 3-dimensional ICE soon, and in some systems, control of the fusion imaging platform is located on the echocardiography machine.Unlike TEE, direct manipulation of the imaging transducer and console control are generally physically separated in space and by the sterile field. One workflow option can have the interventional imager performing catheter manipulation to optimize the transducer position and imaging plane, especially since the same physics principles considered during TEE probe manipulation apply. Unfortunately, current platforms do not allow full console manipulation from the sterile field. Therefore, the interventional imager's skills may be better used at the echo machine rather than at the probe, since 3-dimensional ICE allows for enough digital imaging plane manipulation options to minimize the need for physical probe position changes.TEE imaging is excellent for transcatheter mitral interventions due to the position of the esophagus relative to the left atrium. The mitral valve is typically less than 10 cm from the probe, and when imaged from the left atrial wall opposite from the mitral valve, is well-positioned for flexible imaging of the entire valve using single plane, biplane or 3-dimensional imaging. When the left atrium is massively enlarged, the esophagus is significantly displaced laterally, or when other anatomic abnormalities limit TEE image quality, ICE may provide better intraprocedural imaging .Optimal visualization of the mitral valve is generally achieved by positioning the ICE probe in the left atrium across a trans-septal puncture, by first positioning the tip of the ICE probe near the puncture site and then crossing using anterior flexion and gentle catheter advancement. This is usually done through the same puncture site as the device delivery system, with the main advantages being reduced trauma to the interatrial septum and increased simplicity compared to requiring a second separate puncture. The disadvantages include possible interaction between the delivery system and ICE catheter during delivery system manipulation, which may cause undesired movement of the ICE probe. The close proximity between the ICE probe and delivery system may also cause increased acoustic shadowing if the ICE probe is positioned in such a way that the catheters are in the line of sight between the transducer array and mitral valve. Using two separate punctures may reduce interaction and shadowing from the delivery system, at the cost of slightly increased procedural complexity and increased trauma to the interatrial septum.Once the ICE probe is positioned in the left atrium, the face of the imaging array should be positioned as parallel to the mitral annulus as possible using the appropriate amount of anterior or posterior flexion, although additional adjustments may be needed to avoid or minimize acoustic shadowing from delivery system catheters . Left orAngulation of the asymmetric imaging array relative to the mitral coaptation line is also affected by left or right flexion. To maximize biplane quality for leaflet segment coaptation pair visualization, such as for leaflet grasping in transcatheter edge-to-edge repair, it is best to obtain an intercommissural view at either −45 or +45 degrees so the orthogonal “grasp” or long axis view is taken at +45 or +135 degrees. As described above, this is to overcome the image degradation that occurs perpendicular to the long axis of the ICE probe (at 90 degrees) due to the crystal arrangement of the ICE matrix array. After optimization, good quality biplane, live 3-dimensional and live 3-dimensional multi-planar reconstruction can be performed similarly to TEE imaging .Another window where the mitral valve and subvalvular apparatus are well-visualized is from the right ventricle. After crossing the tricuspid valve, rotation of the catheter to face the left ventricle can provide long and short axis views of the mitral valve from the ventricular perspective.Steering devices through the left atrium may require a different ICE position to better visualize the left atrial free wall, left atrial appendage and pulmonary veins. This can be achieved by posterior flexion of the probe in the left atrium so it is resting almost against the interatrial septum.Other special situations where 3-dimensional ICE can be considered for mitral interventions are in situations where TEE is extremely technically difficult, contraindicated due to anatomical factors , or the risk of general anesthesia and mechanical ventilation is very high or prohibitive . In the TEE image quality during transcatheter tricuspid interventions is variable due to the position of the tricuspid valve relative to the TEE probe. When the atria are dilated, the tricuspid valve may be in the far field for TEE. There may also be significant acoustic shadowing from intracardiac structures and prostheses. Lastly, cardiac chamber dilation often causes esophageal displacement relative to the atrioventricular valves, creating a “horizontal” appearance and further acoustic shadowing of the valve once delivery catheters are placed in the heart. Due to these factors, 3-dimensional ICE has significant advantages over TEE. The right atrial position of the probe places the tricuspid valve in the near field and avoids acoustic shadowing from left-sided structures and the crux of the heart .The tricuspid valve is well-visualized using ICE without significant manipulation. Once the catheter is introduced into the right atrium from the inferior vena cava, the tricuspid valve is immediately visualized in the ICE “home view”. From this position, left or right flexion can be used to optimize angulation of the transducer face relative to the commissures to obtain the best biplane and 3-dimenional imaging, similar to what is described above for the mitral valve .en face view where all leaflets are visualized in short axis (One of the critical TEE views used in transcatheter tricuspid interventions is the ort axis . This isort axis .The interatrial septum is well visualized by ICE after clockwise rotation of the probe from the “home view” to face the interatrial septum. Posterior flexion is helpful to position the probe away from contacting the septum directly to better visualize the fossa . A smallOnce the catheter is positioned, biplane or live 3-dimensional multi-planar reconstruction can be used to evaluate the interatrial septum by reproducing planes similar to those used with TEE image guidance of trans-septal punctures or atrial septal interventions. This includes a bicaval-like view, aortic short axis view and 4-chamber view .In some atrial septal interventions, such as patent foramen ovale closure or simpler atrial septal defect closures, the use of ICE image guidance allows these procedures to be performed without general anesthesia and may reduce procedure-related costs, risks, and length of stay , 9.ICE is being used more and more for left atrial appendage closure and 3-dimensional ICE has some potential advantages. In addition to biplane imaging to evaluate device positioning, 3-dimensional multi-planar reconstruction allows for appendage sizing similar to TEE. Precise trans-septal puncture can be performed using the techniques described above.While the left atrial appendage is well visualized across the interatrial septum from the right atrium, image quality can be improved by crossing into the left atrium across a trans-septal puncture. Positioning the transducer to face the left atrial appendage by using posterior flexion to place it almost on the interatrial septum allows for optimal 2-dimensional, biplane and 3-dimensional image quality .In addition to excellent visualization of the mitral and tricuspid valves as described above, aortic valve imaging planes can also be achieved using 3-dimensional ICE. Clockwise rotation from the home view and adjustment of anterior-posterior flexion such that the probe sits near the tricuspid annulus inferior to the aorta combined with slight leftward flexion can often visualize the aortic valve in short axis plane clearly. Additional short axis and long axis views can also be achieved from within the right ventricle. As a result, closure of mitral, tricuspid and aortic prosthetic paravalvular leaks can be guided by 3-dimensional ICE.3-dimensional ICE guided paravalvular leak closure may have the unique advantage of closer probe proximity to the prosthesis and defect, which may visualize the leak, catheters and wires even more clearly than by TEE. This may be particularly true for aortic paravalvular leaks, which can be challenging to visualize and guide by TEE due to the distance of the aortic valve from the probe and significant acoustic shadowing due to the prosthesis itself. 3-dimensional ICE guidance may also permit the avoidance of general anesthesia to reduce procedural risks and length of hospital stay.There are definite limitations to the current widespread adoption and successful use of 3-dimensional ICE for transcatheter structural interventions. Catheter cost is one of the primary barriers, but imaging platform compatibility and appropriate reimbursement, especially for interventional imaging experts, are also important factors in most centers. The inability to fully replicate all TEE views and some image quality limitations, as described above, are potential procedural limitations. Expertise, training, and workflow optimization are also important factors that will need to be addressed.3-dimensional ICE has a promising role in transcatheter structural heart interventions, especially when TEE imaging is technically challenging, or minimization of sedation requirements is desired. The overall experience of the structural heart community is growing, but ongoing work is needed to refine techniques and workflow to maximize the utility of this imaging modality.


Sodium‐glucose cotransporter (SGLT)‐2 inhibitors have recently been approved for chronic kidney disease (CKD) based on their ability to lower proteinuria and slow CKD progression independent of diabetes status. In diabetic renal disease, modulation of tubuloglomerular feedback (TGF) leading to lower intraglomerular pressure has been postulated as one of the mechanisms of renal protection with SGLT2 inhibition; however, this mechanism has not been sufficiently explored in non‐diabetic CKD. We hypothesized that SGLT2 inhibition exerts renoprotection in CKD through increasing TGF despite normoglycemia. To test this hypothesis, we used an integrative mathematical model of human physiology, HumMod. Stage 3 CKD conditions were simulated by reducing nephron mass which was associated with hypertension, low glomerular filtration rate (GFR) (55 mL/min), hyperfiltration of remnant nephrons, elevated albuminuria (500 mg/day), and minimal levels of urinary glucose (0.02 mmol/L). SGLT2 inhibition was associated with acute reductions in GFR associated with afferent arteriolar vasoconstriction due to TGF. After 12 months, glomerular pressure, nephron damage, and chronic GFR decline were reduced with SGLT2 inhibition with additional SGLT1 inhibitory effects further enhancing these effects. This model supports the use of SGLT2 inhibitors to reduce hyperfiltration in CKD and mitigate renal disease progression, even in the absence of diabetes. ProximaRenal hemodynamics and tubular functions are influenced by physical factors, the sympathetic nervous system, circulating hormones, and TGF. TGF is primarily determined by the delivery of filtered sodium to the macula densa, but also by angiotensin II and ANP (both minor effects) Figure . AdditioPlasma levels of hormones are calculated from rates of secretion/production, clearance rates, and volume of distribution. Key determinants of renin secretion are β‐adrenergic stimulation of juxtaglomerular cells and sodium chloride delivery to the macula densa, the latter influenced by both RSNA and ANP Figure . The pri2.2Bioavailability of empagliflozin in the model was assumed 78% . Glomerular permeability to albumin was also increased to give significant baseline albuminuria (500 mg/day) Table . These cCKD: Low renal function (25% nephrons remaining)CKD + SGLT2i: CKD model with 25 mg empagliflozin/day with only inhibitory capabilities on SGLT2CKD + SGLT1/2i: CKD model with 25 mg empagliflozin/day with inhibitory capabilities on both SGLT1 and SGLT2 activity.After recording 1.5 months of baseline values with 3 high carbohydrate meals per day, responses to 1 year of SGLT2 inhibition were determined for each of the following simulations:Additional simulations determining the effect of changing carbohydrate or salt intake on chronic CKD responses are provided in the supplement  as compared to normal (58%) resulting from suppressed angiotensin II, increased atrial natriuretic peptide, and high renal interstitial fluid pressure  short‐term marker for glomerular pressure and to gauge the beneficial responses to renoprotective treatments such as RAS inhibitors. The current model predictions suggest SGLT2 reduces albuminuria 5%–10% acutely and is sustained for 12 months Figure . ClinicaThe only large randomized trial investigating the impact of SGLT2/1 inhibition (sotagliflozin) in CKD was performed recently in diabetic individuals with or without albuminuria . Mild glycosuria has been noted in CKD patients. Causes and consequences of glycosuria in non‐diabetic CKD is not well studied. Increased fractional excretion of glucose occurs with more advanced stages of CKD due to tubular dysfunction and is associated with greater proteinuria  may need to be included in future models to better understand the cardiovascular responses to SGLT2 inhibition. Third, SGLT2 inhibition may exert additional renal benefit through weight reduction in the obese population, but this weight loss is usually modest (1 kg over 2 years in CKD patients)  and National Institute of General Medical Sciences (U54 GM115428) to JSC and the National Institute of General Medical Sciences (P20 GM104357) to the UMMC Department of Physiology.The authors declare that there are no competing interests.None.Appendix S1:Click here for additional data file.


The purpose of this prospective ultrasound-based pilot study was to identify the most suitable tibial puncture site for intraosseous (IO) access in term and preterm neonates, describe tibial dimensions at this site, and provide anatomical landmarks for rapid localization. We measured the tibial dimensions and distances to anatomical landmarks at puncture sites A  and B , in 40 newborns in four weight groups . Sites were rejected if they fell short of the assumed safety distance to the tibial growth plate of 10 mm. If both A and B were rejected, puncture site C was determined sonographically at the maximum tibial diameter while maintaining the safety distance. Puncture site A violated the safety distance in 53% and 85%  and puncture site B in 38% and 33%. In newborns weighing 3000–4000 g, at median (IQR), the most suitable puncture site at the proximal tibia was 13.0 mm (12.0–15.8) distal to the tuberosity and 6.0 mm (4.0–8.0) medial to the anterior rim of the tibia. The median (IQR) diameters at this site were 8.3 mm (7.9–9.1) (transverse) and 9.2 mm (8.9–9.8) (anterior–posterior). The diameters increased significantly with increasing weight.  Conclusion: This study adds concise, practical information on the implementation of IO access in neonatal patients: the tibial dimensions in newborns in four different weight groups and initial data on anatomical landmarks to easily locate the IO puncture site. The results may help implement IO access in newborns more safely. During the rare event of newborn resuscitation, in addition to effective ventilation of the lung and chest compressions, venous access must be established rapidly to administer lifesaving drugs. In 2021, the guidelines for Newborn Life Support of the European Resuscitation Council included intraosseous (IO) access as a reasonable alternative to the primary method via umbilical vein catheters for emergency vascular access [In pediatric patients aged less than 1 year, the recommended tibial puncture sites are 10 to 20 mm (or two finger-widths) distal to the tibial tuberosity at the proximal tibia and 10 to 20 mm proximal to the malleolus medialis at the distal tibia. This relatively broad recommendation was suggested to be adjusted to 10-mm distance at the proximal tibia for the age group of newborns . HoweverTo date, several studies and case reports describe the successful use of IO access in newborns –13. HoweThe aims of our study were to (1) identify the most suitable puncture site at the proximal and distal tibia for IO access in term and preterm neonates, (2) define distances to anatomical landmarks to easily locate the puncture site, and (3) provide the dimensions of the tibial bone at these sites.As there is limited knowledge about the exact tibial bone growth rate at the IO puncture site in different gestational age or body weight groups , 17, thiAfter obtaining written informed consent from the parents, the tibia of the newborn was measured using a specially developed standardized ultrasound protocol Fig. , and, inAll measurements were performed by two study team members (SM or TH) using a mobile ultrasound device  and a linear probe .The aim was to identify the most suitable puncture site with the largest transverse  and anterior–posterior  tibial diameters and at least 10-mm safety distance from the tibial growth plate .We measured the proximal and distal tibial IO puncture sites according to a standardized acquisition protocol Fig.  and detePuncture site A: 10 mm distal to the tibial tuberosity, 10 mm proximal to the medial malleolus of the tibia (according to the lower border of the pediatric recommendation).Puncture site B: chosen by a pediatrician by palpation and subjective assessment.Puncture site C: sonographically determined in case puncture sites A and B violated the safe distance of 10 mm from the tibial growth plate.One of the three puncture sites was designated as “most suitable” and used for further calculation; if either A or B met the safety distance, this site was used as “most suitable.” If A and B both met the safety distance, the site with the larger tibial diameter was considered. If neither A nor B met the safety distance, C was used. Measurements that exceeded 15-mm distance from the growth plate were further omitted accounting for the fact that the tibial diameter tapers with increasing distance from the growth plate resulting in smaller diameters.The tibial tuberosity, inferior edge of the patella, anterior rim of the tibia, and medial malleolus were used as anatomical landmarks. The distance between the most suitable IO puncture site and these landmarks was measured on the skin surface.Tibial diameters were determined using ultrasonography Fig. . The trap < 0.05).Statistical analysis was performed using R-Studio v4.1.2 , employing non-parametric Kruskal–Wallis rank-sum tests. Post hoc analyses for pairwise comparisons (Conover-Iman’s test) among the weight groups were performed with Bonferroni correction for multiple comparisons if the null-hypothesis, i.e., “all weight groups are equal,” was rejected by the Kruskal–Wallis test .In total, data of 40 term and preterm infants  were collected in this study, with ten subjects in each body weight group. The patient demographics are shown in Table Puncture site A  violated the safety distance to the growth plate in 53% and 85% of cases, at the proximal and distal tibia, respectively. Puncture site B (subjective assessment by pediatricians) violated the safety distance by 38% and 33% . In 23% of cases, the safety distance was violated at both puncture sites A and B at the proximal tibia and in 38% of cases at the distal tibia. In these cases, puncture site C was identified using ultrasonography.The number of puncture sites A and B that met the safety distance, as well as the distance to the growth plate, increased with body weight, exempting puncture site A at the distal tibia, where the opposite trend was observed Fig. .Fig. 3MeAnatomical landmarks can be used as guidance to locate the most suitable puncture sites. In the weight group with infants of 3000–4000 g body weight, at a median , the most suitable puncture site was located 13.0 mm 12.0–15.8) distally from the tibial tuberosity and 6.0 mm (4.0–8.0) medially from the anterior rim of the tibia. Details for the other weight groups and distal tibia as well as statistics are given in Table .0–15.8 dp = 0.60; distance to lower edge of patella, p = 0.93). At the distal tibia, the distance from the landmarks malleolus medialis and anterior tibial rim to the most suitable puncture site was dependent on the weight group (p < 0.001 and p = 0.002), with larger landmark distances to the puncture site in groups with higher body weight.At the proximal tibia, there was no significant difference in the distances from landmarks to puncture sites according to weight groups  transverse diameter at the most suitable puncture site of the proximal tibia was 8.3 mm (7.9–9.1) and the anterior–posterior diameter was 9.2 mm (8.9–9.8). At the distal tibia, we measured a median (IQR) transverse and anterior–posterior diameter of 6.8 mm (5.7–7.7) and 10.8 mm (10.1–11.6), respectively. Details of all weight groups are shown in Table The aims of this ultrasound-based pilot study were to (1) determine the most suitable puncture site for IO access in the tibia of term and preterm newborns, (2) identify anatomical landmarks to determine the most suitable puncture site, and (3) measure the tibial dimensions at the IO puncture site.To achieve high success rates in IO implementation and avoid complications, it is essential to accurately determine and locate the most suitable IO puncture site with the largest possible dimensions and diameters , 12. HowIn this study, to avoid accidental injury to the growth plate, we assumed the safe distance between the growth plate and the IO puncture site to be 10 mm. Interestingly, when following the lower limit of the aforementioned pediatric recommendation , this safety distance was often not met . This was similar for puncture site B, which was subjectively chosen by the pediatrician . This finding is in contrast to that of Boon et al. , who fouHowever, it is important to note that the 10-mm safety distance to the growth plate was chosen as a purely pragmatic approach in the design of this study. To the best of our knowledge, there is no clear recommendation as to how large the safety distance to the growth plate should be. There are no clear data on the long-term consequences in cases of accidental perforation of the growth plate. Claudet et al.  found noIO access is an extremely rare event even for experienced neonatologists. Therefore, it may be difficult to find the most suitable puncture site, especially in demanding, life-threatening emergencies. Specific anatomical landmarks to facilitate the localization of IO puncture sites would be beneficial; however, to our knowledge, so far, there is no study that has yet described clear landmarks. Therefore, we intended to provide initial data to fill this gap.According to the results obtained in this pilot study, in a newborn with a body weight of 3000–4000 g, a measured median distance of 13.0 mm distal to the tibial tuberosity and 6.0 mm medial to the anterior rim of the tibia would lead to the most suitable puncture site for IO access at the proximal tibia. In addition, we provided data for newborns and preterms with lower body weights down to less than 1000 g. Interestingly, at the proximal tibia, these distances were similar across all weight groups and did not differ significantly in the weight groups. Certainly, these preliminary findings must be confirmed in future studies. Also, it must be emphasized that there is not yet sufficient data, and therefore, no recommendations can be made for the use of IO access in preterm infants. Manufacturers of IO needles also recommend a minimum weight for the establishment of IO access in newborns and preterm infants, which should be taken into account by providers.It is noteworthy that, depending on the amount of fat tissue, palpability and clear identification of the tibial tuberosity were difficult in some cases. In addition, the tuberosity presents itself more as a plateau when palpated than as a clear demarcation line, which impedes its precise localization. In contrast, in most cases, the lower edge of the patella was palpated as a clear line, and therefore might represent an easier to palpate alternative landmark to find the most suitable IO puncture site. Additionally, the distance of the lower edge of the patella to the most suitable puncture site showed relatively low variation in our results.To our knowledge, this is the first study to provide clinical guidance for anatomical landmarks based on ultrasound validation to determine the most suitable IO access in newborns. Nonetheless, owing to the pilot nature of this study and the small sample size in each weight group, future studies are warranted to derive recommendations for optimal IO access based on distances to anatomical landmarks in term and preterm neonates.For the successful implementation of IO access in newborns, it is important to be aware of the expansion of tibia at the puncture site in order to safely place the IO needle in the medullary cavity , 7. TherAs expected, large differences between the weight groups were found, such that the diameters at the puncture sites were larger with increasing weight of the infants. The mean (median) diameter of the proximal tibia at the most suitable puncture site was 8.4 mm 8.3) (transverse) and 9.2 mm (9.2) (anterior–posterior) in the 3000–4000 g weight group. These diameters are slightly larger than those reported by Suominen et al.   and Tond (transveAlso, future studies are warranted to analyze tibial diameters at different tibial heights to identify the optimal puncture sites as well as long-term consequences in case of injury to the growth plate.We chose ultrasound as the method of investigation because it was most appropriate for term and preterm infants, some of whom required intensive care. Other methods, such as X-ray or computed tomography, may have yielded other or more precise results.Furthermore, the relatively small number of patients in this pilot study does not allow us to establish normative values for the optimal site of intraosseous access in neonates. Nonetheless, it presents a valuable basis for further research on IO-puncture sites in neonates, which is essential for optimal and safe performance in emergency situations.Finally, it must be emphasized that this study only investigated the tibia as an option for IO access, which seems to be the most commonly selected site for IO access in neonatal and pediatric resuscitation , 11, to Knowledge of the optimal puncture site for IO access and its anatomical characteristics is crucial for the successful implementation of IO access in newborns. With this pilot study, we were able to present initial data for the creation of anatomical landmarks for rapid localization of the most suitable puncture site in neonates. In addition, we provided data on the dimensions of the tibia at the IO puncture site in the different weight groups. The findings of this study may help implement IO access more safely in newborns.


Influenza B virus (IBV) is one of the two major types of influenza viruses that circulate each year. Unlike influenza A viruses, IBV does not harbor pandemic potential due to its lack of historical circulation in non-human hosts. Many studies and reviews have highlighted important factors for host determination of influenza A viruses. However, much less is known about the factors driving IBV replication in humans. We hypothesize that similar factors influence the host restriction of IBV. Here, we compile and review the current understanding of host factors crucial for the various stages of the IBV viral replication cycle. While we discovered the research in this area of IBV is limited, we review known host factors that may indicate possible host restriction of IBV to humans. These factors include the IBV hemagglutinin (HA) protein, host nuclear factors, and viral immune evasion proteins. Our review frames the current understanding of IBV adaptations to replication in humans. However, this review is limited by the amount of research previously completed on IBV host determinants and would benefit from additional future research in this area. Severe influenza infections in humans are typically caused by two influenza virus types: influenza A viruses (IAV) and influenza B viruses (IBV). These infections are suspected to cause up to 5 million severe cases of influenza disease annually . In the Throughout recorded history, IAV has been responsible for four major pandemic outbreaks, in 1918, 1957, 1968, and 2009. These pandemics were caused by the H1N1, H2N2, H3N2, and H1N1 subtypes, respectively . The strDue to shared structural and replicative properties with IAV, we hypothesized that the distinct difference in host range between IBV and IAV is unlikely to be due to chance alone. Viral and host factors important to IAV host determination have been reviewed previously ,14. HoweIn the initial stages of infection, IBV must bind to sialic acid residues on the surface of the cells lining the respiratory tract. Receptor binding is facilitated by the IBV hemagglutinin protein (B-HA). This receptor-binding interaction is crucial for successful viral infection and plays a major role in host specificity of IAV ,16,17. TTo facilitate membrane fusion between the viral envelope and the host endosome after receptor interaction, B-HA must have been enzymatically activated . While oOutside of receptor binding and enzymatic activation, Laporte, et al. has shown that the physical environment of the human upper respiratory tract plays a key role in facilitating IBV infection. Interestingly, the pH required for efficient B-HA activation and membrane fusion was lower for IBV strains than for avian IAV (5.4–5.6 for IBV compared to 5.7–5.8 for avian IAV) . AdditioThe receptor abundance in the human respiratory tract, the presence of enzymes capable of B-HA activation, and the optimal physical environment for IBV infection suggest possible host restriction of IBV replication to humans over non-human hosts. A summary of these factors can be seen in Influenza viruses belong to a group of RNA viruses that replicate their genome in the nucleus rather than the cytoplasm ,48 despiOnce in the nucleus, B-Pol transcribes the viral RNA to initiate the viral replication cycle. Human acidic nuclear phosphoproteins (ANP32) are crucial for influenza RdRp activity ,55,56,57Again, few studies describe the interactions of IBV with host nuclear factors for the replication process. As mentioned briefly, the interactions of IBV with human importin-α focus on binding affinities but do not investigate how non-human homologs impact viral replication. These studies also fail to fully establish a mechanism of nuclear transport across the nuclear envelope. To support importin-α acting as a host restriction factor, future studies should assess the ability of different species’ importin-α to support IBV replication. This would provide evidence regarding to what extent IBV can utilize other species’ importin-α for nuclear transport to replicate the viral genome and express the viral proteins. While the importance of human ANP32 proteins was shown more clearly for IBV replication than importin-α, only avian ANP32 proteins were used to compare more than the two primary forms of ANP32 proteins. A major limitation to the studies showing ANP32 proteins supporting B-Pol activity was the absence of a comparison between the ability of host ANP32 proteins to support viral replication. Only B-Pol activity was measured in a minigenome reporter assay . AnotherA critical relationship between a host and pathogen is immune evasion. Influenza viruses specifically encode proteins to aid in immune evasion and utilize proteins with other viral functions to assist in evasion. A major difference between IAV and IBV in immune evasion is the lack of alternative polymerase protein products encoded in the PB1 and PA gene segments. IAV PB1-F2  and PA-XThe interferon (IFN) and interferon-stimulated gene (ISG) signaling pathways are a primary defense for the innate immune system in humans. During influenza infection, host cells produce IFN to activate host ISGs and limit total gene expression . This imIn addition to avoiding innate immune detection within the cell, the virus must also evade any adaptive immunity to infected cells. This recognition can come from cytotoxic T lymphocytes (CTLs). One way the virus attempts to avoid CTL recognition is through the downregulation of MHC Class I surface expression in infected cells. After IBV infection, cell lines stably expressing different HLA-alleles showed significantly decreased MHC Class I surface expression compared to IAV infection . AnotherWe hypothesized that a virus with a limited host range such as IBV will be effective at evading its primary host’s immunity but not other host immunities. This would be due to the absence of viral responses to immune pressures exerted by non-human hosts, limiting virus replication. We also hypothesize that a virus with multiple mechanisms of immune evasion is more likely to have an expanded host range. The literature characterizing the B-NS1 interaction with human ISG15 supports our first hypothesis. As described above, B-NS1 is only able to rescue virus replication in the presence of human ISG15, while ISG15s from non-human species are able to inhibit replication in the presence of B-NS1. This suggests that IBV could be unable to successfully evade an aspect of non-human host immunity that prevents viral replication and spread within that host. We expect this species-specific targeting of ISG15, combined with other immune evasion mechanisms, plays a key role in promoting IBV circulation in humans while also potentially restricting replication in other non-human hosts.Other immune evasion mechanisms have been characterized for IBV. Protein kinase R (PKR) ,88, humaThe lack of an established non-human host presents a major difference between influenza B viruses and influenza A viruses. With the exception of isolated mammalian detection , no majoThe most extensively interrogated, possible IBV host restriction factor is the B-HA protein. The interaction between viral protein and host receptor is a crucial determinant of viral entry into susceptible cells. Unsurprisingly, host receptor characteristics likely shape whether or not that host can be infected. These characteristics are summarized in Possible host restriction factors outside of B-HA are not as well understood. To determine if a host restriction phenomenon has been observed during IBV genome replication, we considered host nuclear factors. The importin-α protein family has been identified as a critical factor for transporting the vRNPs from the cytoplasm into the nucleus ,52. HoweIBV has also evolved several mechanisms to escape human immune recognition. B-NS1 drives multiple mechanisms that obstruct the human IFN response ,74. TargA major theme to this research topic is the relative lack of studies investigating the possible IBV host restriction factors. Numerous studies have identified host factors or host–viral interactions critical for efficient IBV viral replication. However, these studies often fail to compare the human factors with non-human homologs, leading to a gap in our knowledge of why IBV only circulates in humans. Another potential avenue to discovery of IBV host restriction factors is considering the restriction factors of other types of influenza viruses. Factors that limit IAV, especially avian IAV, have been comprehensively reviewed ,14,93,94Two other influenza types have also been discovered and circulate in mammals. Influenza C viruses (ICV) are distantly related to IBV and primarily infect humans . InfluenDespite an established host restriction environment for influenza viruses, host spillover events are detected. The most well-known examples are the pandemic spillover events from past influenza pandemics . HoweverA potential piece of non-molecular evidence supporting strong host restriction factors for IBV circulation is the possible extinction of the Yamagata-like lineage. The 2019–2020 flu season was predominantly characterized by the emergence of the new Victoria-like clade V1A.3 ,46. ThisBased on our review of the available literature, there appears to be some evidence of IBV adaptation to human circulation throughout different stages of the replication cycle. Researchers have identified crucial interactions during receptor binding, genome replication, and immune evasion between viral and human factors that permit optimal virus replication to occur. The fact that these interactions occur throughout the virus replication cycle and not just during viral entry suggests that there is likely an important relationship between the human host and IBV replication. Many of these interactions require more comparison studies to investigate the impact that the factors from different host species have on IBV replication. This would help support the role of these factors as IBV host restriction factors. However, logically analyzing the available evidence suggests these adaptations could limit the ability of IBV to circulate in hon-human hosts. A better understanding of these interactions could unlock new information about how influenza viruses are restricted to their hosts. This information could then be leveraged as novel, specific antiviral targets in the future.


This study attempts to elucidate the prevalence and genetic factors associated with four angular hoof deviations in the Pura Raza Española horse: splay-footed forelimb (SFF), pigeon-toed forelimb (PTF), splay-footed rear limb (SFR), and pigeon-toed rear limb (PTR). From a total of 51,134 horses evaluated, only 15.75% did not have any of the four investigated angular hoof defects, while the rest presented varying degrees of defect. We identified the factors influencing these defects, including age, inbreeding coefficient, sex, and birth stud size, using a Bayesian multivariate animal model with the BLUPF90 software. The heritability estimates ranged from 0.11 (SFR) to 0.31 (PTR) for model A (three categories.), indicating the genetic influences of varying categories; and ranged from 0.11 (SFR) to 0.29 (PTR) for model B (two categories). Additionally, the study explored the genetic correlations between these defects and their relationship with the diameter of the superficial digital flexor tendon (SDFT) and the proportionality index (PI). The findings revealed that diameter of the SDFT is strongly correlated with inward-toe conditions (PTF and PTR), while PI is associated with outward defects (SFF and SFR). These results provide valuable insights into angular hoof deviations in PRE horses, shedding light on their prevalence, genetic factors, and associations with other anatomical features, which can have general implications for a horse’s health and performance.P≠0 ≥ 0.95), while PI is associated with outward toe defects .Abnormalities in hoof shape are usually connected with limb conformation defects. The role of angular hoof deviations is important for longevity in sports competitions and is increasingly recognized as a factor associated with lameness in performance horses. In this paper, we measured the prevalence of four defects related to the angulation of the hoof in the Pura Raza Española horse (PRE): splay-footed forelimb (SFF), pigeon-toed forelimb (PTF), splay-footed rear limb (SFR), and pigeon-toed rear limb (PTR). A total of 51,134 animals were studied, of which only 15.75% did not have any of the four angular hoof defects investigated, while 26.61%, 23.76%, 79.53%, and 3.86% presented SFF, PTF, SFR, and PTR, respectively. Angular defects were evaluated using two different models; model A was a linear scale composed of three categories, where 0 corresponded to the absence of defects, 1 to a minor presence of the defect and 2 to the highest degree of the defect. Model B was composed of two categories, where 0 corresponded to the absence of defects and 1 to the presence of defects, joining classes 1 and 2. We measured the factors influencing the appearance of these defects: age, inbreeding coefficient, sex, and birth stud size. The heritability of each defect was also estimated using a multivariate animal model, using the Gibbsf90+ software from the BLUPF90 family, resulting in heritability estimates of 0.18 (s.d. = 0.009), 0.20 (s.d. = 0.010), 0.11 (s.d. = 0.009), and 0.31 (s.d. = 0.010) for SFF, PTF, SFR, and PTR defects, respectively, for model A, and 0.17 (s.d. = 0.008), 0.19 (s.d. = 0.009), 0.11 (s.d. = 0.009), and 0.29 (s.d. = 0.009) for SFF, PTF, SFR, and PTR defects, respectively, for model B. Finally, the genetic correlation between the diameter of the superficial digital flexor tendon (SDFT) and the proportionality index (PI) in relation to the higher or lower prevalence of the defects was analyzed. We concluded that diameter of SDFT development is strongly correlated with inward toe conditions (PTF, PTR;  The hoof structure plays a critical role in locomotion, weight distribution, and shock absorption during movement in equines , and disAngular hoof deviations encompass a spectrum of abnormalities that involve the alignment and orientation of a horse’s hooves in relation to the limb axis. These deviations impact the biomechanics of the horse’s limbs and, consequently, influence the horse’s performance in various activities, including work and sports competitions, or simply its ability to move comfortably without pain. These areas’ defects are associated with a series of factors, such as injuries, inflammation, bio-mechanical imbalances, abnormal development during the horse’s growth, or even genetic factors. The main consequence is the effect on the horse’s ability to move correctly ,5. ThrouIn this context, although all the above-mentioned risks produce themselves financial losses and a lower quality of life for the horse, from a population management point of view, knowledge about the genetic implications is of great interest. The identification and study of limb defects are essential for implementing the appropriate selection measures to reduce their incidence and improve the health and athletic performance of the breed, especially when considering sport competition horses such as the Pura Raza Española (PRE) horse. The PRE is a native Spanish breed, one of the oldest in Europe and one of the most important at the international level. This breed has gained international recognition in the field of Classical Dressage, taking part annually in a significant number of competitions, both in Spain and worldwide . In geneThis study aims to provide a more comprehensive understanding of the factors affecting hoof health in PRE horses, with potential implications for the breeding program, management practices, and overall well-being of this equine breed. In this study, the incidence of hoof defects in the PRE horse population was estimated and the potential influencing risk factors in the presentation of angular hoof deviation were analyzed. Additionally, genetic parameters related to these defects were examined to determine if there is a genetic basis contributing to their occurrence. Finally, the relationship between the presence of hoof defects and morphological variables, specifically the diameter of the superficial digital flexor tendon (SDFT) and the proportionality index (PI), were explored.In our study, we analyzed hoof conformational data from 51,134 Pura Raza Española (PRE) horses . The sample encompassed a variety of hoof conformational defects : hoof deDefects of the ‘splay-footed forelimb’ (SFF) and ‘pigeon-toed forelimb’ (PTF). These defects are related to the direction of the front hooves when seen from the front. An individual exhibits these defects when the hoof points either inwards or outwards, respectively, in relation to the vertical plumb line, as measured from the outer edge of the shoulder joint and the humero-cubital joint to the ground.Defects of the ‘splay-footed rear’ limb (SFR) and ‘pigeon-toed rear limb’ (PTR). These defects are related to the direction of the rear hooves when seen from the back. An individual has an SFR or PTR defect when the hoof tips either inwards or outwards, respectively, in relation to the vertical plumb line, as measured from the point of the hip and tibia-fibula joint to the ground, when seen from the rear.The four analyzed defects were as follows:The estimation of genetic parameters was performed with two different models:For model A, the four conformational hoof defects were recorded on a linear scale from 0 to 2. Category 0: represents a lack of the defect, the hooves are vertical, or slightly turned outwards (SFF or SFR) or inwards (PTF or PTR), with, at most, 5 degrees of angulation. Category 1: a minor presence of the defect, the hooves are turned outwards (SFF or SFR) or inwards (PTF or PTR) between 5 and 20 degrees of angulation. Category 2: the most significant level of the defect, the hooves are turned outwards (SFF or SFR) or inwards (PTF or PTR) with more than 20 degrees of angulation.For model B, the four conformational hoof defects were recorded as a dichotomous model. Category 0: represents a lack of the defect, the hooves are vertical, or slightly turned outwards (SFF or SFR) or inwards (PTF or PTR), with, at most, 5 degrees of angulation. Category 1: presence of the defect, the hooves are turned outwards (SFF or SFR) or inwards (PTF or PTR), with more than 5 degrees of angulation.In PRE horses, SFF and SFR defects are considered to be severe when they are in category 2, and PTR and PTF defects are considered to be very severe when they are in category 2. When a PRE horse, in any part of its body, has more than 2 very severe defects or more than 4 severe defects, the horse cannot be registered as a breeder of the PRE breed.The covariates analyzed were the evaluation age  and the classical inbreeding coefficient . The F value, following Wright , definedAdditionally, heritabilities and genetic correlations between the hoof defects and two morphological variables, the diameter of the superficial digital flexor tendon (SDFT), and the proportionality index (PI) were analyzed. The SDFT extends from the elbow region to the distal interphalangeal joint in the forelimb or from the hock region in the hind limb. The diameter of the SDFT was measured on a linear scale from 1 to 9, depending on the thickness of the tendon of the four limbs as a whole, where 1 is very thin and 9 is highly developed (larger diameter); the evaluation is unique, grouping, in a single evaluation, both forelimb and rear limb diameter. This tendon is a fundamental part of the connective system that allows for the function of hoof flexion in the horse’s limbs. The PI was defined as the height at the withers * 100 divided by the scapular-ischial length (distance of the straight segment from the union of the scapular-humerus joint to the point of the buttock). The PI is a variable widely used in equine breed characterization , and alsA multivariate Generalized Non-linear Model (GLZ), with a multinomial logit distribution, was used to examine associations between the conformational hoof defects and their potential factors. All the statistical analyses were conducted using Statistica 11 for Windows software .y is the vector of observations, X is the incidence matrix of fixed effects, Z is the incidence matrix of the animal genetic effect, b is the vector of fixed effects, u is the vector of the direct animal genetic effect, e is the vector of residuals, I is an identity matrix, and A is the numerator relationship matrix.For the genetic evaluations, the genealogical information was obtained from the PRE StudBook managed by ANCCE , ensurinThe genetic parameters were estimated using the Blupf90 software . A BayesThe basic statistical results for angular hoof deviations in the PRE horses are shown in The percentage of affected animals (category 1 or 2) for each hoof defect can be observed in In the analysis of angular hoof deviations in PRE horses, multiple effects were evaluated, including age, inbreeding, sex, and birth stud size . A GenerThe evolution of the four defects studied in the PRE horses over the last 20 years based on the horses’ birth year is shown in The variance components and heritability values for the four hoof defects studied in PRE horses are shown in P≠0 ≥ 0.95). On the other hand, for model B, the genetic correlations between SFF-PTR, SFF-SFR, and PTF-PTR were positive, with values of 0.14 (s.d. = 0.031), 0.35 (s.d. = 0.054) and 0.30 (s.d. = 0.030) for the three genetics correlations with a P≠0 = 1. The genetic correlation between PTF-SFR was small, with a value of −0.05 (s.d. = 0.055), and not different from zero, as indicated by the symmetric HPD at 95% that included zero (ranged from −0.053 to 0.159) and the low probability of being different from zero (P≠0 = 0.17).The genetic correlations among the four hoof defects are presented in P≠0 = 1.00) In model B, for the SFF and SFR defects, there was no evidence of a relationship between these factors .P≠0 = 0.87 in model A and P≠0 = 0.54 in model B). In model B, for the SFR defects, there was no evidence of a relationship between these factors, not different from zero, as indicated by its symmetric HPD at 95% that included zero and the probability to be lesser than zero (P≠0 = 0.94).Regarding the proportionality index (PI), opposite correlations were observed compared to the SDFT. For model A, the defects SFF and SFR displayed low to moderate positive correlations of 0.212 (s.d. = 0.030) and 0.124 (s.d. = 0.041), respectively. For model B, the defect SFF displayed low to moderate positive correlation of 0.222 (s.d. = 0.031). In both models, for the PTF defect, there was no evidence of a relationship between these factors, not different from zero, as indicated by the symmetric HPD at 95% that included zero and the low probability to be lesser than zero  in the PRE horses. The SFR defect exhibited the highest mean value and a wider confidence interval, indicating greater data dispersion compared to the PTR defect, which had the lowest mean value and the smallest confidence interval . These iIn the PRE horse, the outward deviation hoof defect can vary in severity and may appear in the forelimbs and/or rear limbs, although it is more common in the rear limbs (SFR) . Horses,In each of the four hoof deviation defects, categories 1 and 2 represented the individuals affected, with significantly different percentages being observed among the defects . This inTo achieve an accurate, comprehensive understanding of the patterns in hoof deviation defects, we need to consider that both environmental and genetic factors can influence the occurrence and severity of conformational defects. Although most widely published works have addressed the clinical perspective, no detailed genetic analyses for hoof deviation defects in PRE horses have been extensively conducted until now. In our study, we examined not only the influence of sex and age, but also the individual coefficient of inbreeding and the breeding stud size . OverallHoof variations depend not only on the constant movements made during work  and the Regarding sex, males exhibited a higher incidence of hoof deviation defects compared to females, which aligns with the findings in the lameness study conducted by Ross . This seAn association between the size of the breeding stud and the percentage of affected horses was also observed. Large-scale birth studs primarily focus on breeding, while smaller and medium-sized ones are mainly dedicated to competition. This distinction is crucial, because the type of livestock management significantly influences aspects such as the type of exercise horses undergo, the management regimen, the intensity of training, and other related factors. In this context, it has been documented that foals raised in large herds with extensive management may develop limb conformation irregularities due to uneven load patterns during grazing , as horsRegarding the evolution of defects related to consanguinity levels b, it is Conformation defects have been evaluated in numerous equine breeds using a linear scale that usually ranges from 5 classes ,58 or 9–Regarding the genetic correlations between different hoof deviation defects, our results support that different sets of genes are involved in coordinating hoof deviation defects, depending on whether they occur in an outward or inward position . In a stThe equine limb behaves like a large system of springs due to the extension of the metacarpophalangeal joint and the elastic recoil in tendons ,64; specOn the other hand, the PI in horses seems to be a determining factor that influences movement and, consequently, plays an essential role in maintaining proper balance and hoof conformation . This peIt is possible that there may be limitations in this work, as it was not possible to stan-dardize the age of all the animals since the animals were not evaluated at the same age. Additionally, the presence of animals assessed both with and without horseshoes, as well as their participation in competitions, could have introduced biases in the assessment of angular defects, as footwear and exercise can impact the correction of these issues. Another limitation of phenotyping in the study is the assessment by a single evaluator, rather than using consensus from multiple evaluators for each horse, and variability between horses since there were different single evaluators across the large dataset. Despite this, this work had a significant representation of active PRE horses and it demonstrated that the angular hoof deviations affect limb biomechanics, influencing the horse’s ability to perform, which may also have genetic implications, justifying the implementation of sui-table selection measures to reduce their incidence and improve both the breed and equine sports performance. All these findings emphasize the importance of breeding programs that not only focus on the quality of movements and functionality in dressage, but also place a fundamental emphasis on conformational defects of the horses’ limbs.A high prevalence of angular hoof defects was found in the PRE population, with a higher occurrence in males and significant variations with an increasing inbreeding coe-fficient and breeder herd size. It was demonstrated that genetic factors play a role in the occurrence of these defects, with moderate heritability values. Furthermore, we observed genetic correlations between the defects analyzed, pointing to the existence of specific genes related to the presence of particular defects. Our study also underscored the signi-ficance of taking into account factors such as the proportionality index (PI) and development of the superficial digital flexor tendon (SDFT). It revealed correlations between these factors and the direction of angular hoof defects, with the SDFT being associated with inward defects and the PI with outward defects. Breeders and veterinarians should take into account, when organizing the mating of the stud, the genetic values of the breeding animals for each of the angular hoof deviation defects , along with the genetic values of the rest of the morphological defects of the breed, both in the limbs and other areas of the body. From a reproductive perspective, breeders should prioritize horses whose genetic values, for each of the conformation defects analyzed in the breed , have a high probability of not being transmitted to the offspring. Well-organized horse pairings based on the genetic value of the offspring will reduce defects in the population.