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Abstract Background Staphylococcus aureus (SA) is a leading cause of infectious diseases in sports teams. In recent decades, community-associated SA (CA-SA) strains have emerged worldwide and have been responsible for outbreaks in sports teams. There are very few data on the prevalence of these strains in France, and none on the carriage among athletes. Methods We conducted a cross-sectional study to determine the SA carriage proportion among athletes practicing sports at risk for CA-SA infection in a French county, and determined the methicillin-resistant and/or CA-SA proportion. We also analyzed SA carriage according to risks factors and studied the SA clonality in a sample of our population. Results We included 300 athletes; SA carriage proportion was 61% (n = 183) and one was MRSA carrier (0.33%). The MRSA strain belonged to the clonal complex ST5. None of the strain produced Panton Valentine Leucocidin, and we did not find clonal distribution within the teams. Interestingly, we found a high throat-only carriage (n = 57), 31.1% of the SA carriers. Conclusion We found a high SA carriage with a local epidemiology quite different than that reported in a similar population in the USA. Further studies on SA carriage should include throat sampling. Trial registration The approved protocol was registered on ClinicalTrial.gov , NCT01148485.

Aims: Benralizumab, a humanized, afucosylated monoclonal antibody against the interleukin 5 receptor, α subunit, causes rapid depletion of eosinophils by antibody-dependent cellular cytotoxicity. We investigated the pharmacokinetic and pharmacodynamic effects of benralizumab in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) from the phase III OSTRO trial., Methods: Patients received a placebo or 30 mg of benralizumab by subcutaneous injection every 8 weeks (first three doses every 4 weeks) to week 48; a subset of patients continued in an extended follow-up period to assess treatment durability to week 80. Serum benralizumab concentrations and blood eosinophil and basophil counts were assessed to week 80. Biomarker assessments were performed on nasal polyp tissue biopsies at week 56 and nasal lining fluid at weeks 24 and 56 to examine changes in immune cells and inflammatory mediators., Results: Among 185 patients in this analysis, 93 received benralizumab. Serum benralizumab concentrations reached a steady state by week 24 (median concentration 385.52 ng mL -1 ); blood eosinophils were almost fully depleted and blood basophils were reduced between weeks 16 and 56. Nasal polyp tissue eosinophils decreased with benralizumab from 57.6 cells mm -2 at baseline to 0 cells mm -2 at week 56 (P < .001 vs placebo), and tissue mast cells were numerically reduced. In nasal lining fluid, eosinophil-derived neurotoxin was significantly reduced at weeks 24 and 56 (P < .001) and interleukin-17 at week 56 (P < .05) with benralizumab., Conclusion: Benralizumab treatment led to rapid, sustained, nearly complete depletion of eosinophils from blood and nasal polyp tissue in patients with CRSwNP., (© 2024 AstraZeneca. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-β type II receptor, bolstering its activity in the TGF-β-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-β-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

Objective: Lupus nephritis (LN) is diagnosed by biopsy, but longitudinal monitoring assessment methods are needed. Here, in this preliminary and hypothesis-generating study, we evaluate the potential for using urine proteomics as a non-invasive method to monitor disease activity and damage. Urinary biomarkers were identified and used to develop two novel algorithms that were used to predict LN activity and chronicity., Methods: Baseline urine samples were collected for four cohorts (healthy donors (HDs, n=18), LN (n=42), SLE (n=17) or non-LN kidney disease biopsy control (n=9)), and over 1 year for patients with LN (n=42). Baseline kidney biopsies were available for the LN (n=46) and biopsy control groups (n=9). High-throughput proteomics platforms were used to identify urinary analytes ≥1.5 SD from HD means, which were subjected to stepwise, univariate and multivariate logistic regression modelling to develop predictive algorithms for National Institutes of Health Activity Index (NIH-AI)/National Institutes of Health Chronicity Index (NIH-CI) scores. Kidney biopsies were analysed for macrophage and neutrophil markers using immunohistochemistry (IHC)., Results: In total, 112 urine analytes were identified from LN, SLE and biopsy control patients as both quantifiable and overexpressed compared with HDs. Regression analysis identified proteins associated with the NIH-AI (n=30) and NIH-CI (n=26), with four analytes common to both groups, demonstrating a difference in the mechanisms associated with NIH-AI and NIH-CI. Pathway analysis of the NIH-AI and NIH-CI analytes identified granulocyte-associated and macrophage-associated pathways, and the presence of these cells was confirmed by IHC in kidney biopsies. Four markers each for the NIH-AI and NIH-CI were identified and used in the predictive algorithms. The NIH-AI algorithm sensitivity and specificity were both 93% with a false-positive rate (FPR) of 7%. The NIH-CI algorithm sensitivity was 88%, specificity 96% and FPR 4%. The accuracy for both models was 93%., Conclusions: Longitudinal predictions suggested that patients with baseline NIH-AI scores of ≥8 were most sensitive to improvement over 6-12 months. Viable approaches such as this may enable the use of urine samples to monitor LN over time., Competing Interests: Competing interests: AA, LZ and JAC were formerly employed by, and may hold stock in, AstraZeneca. DS, DC and WIW are currently employed by, and own stock in, AstraZeneca. ABF, JC, MB and SSL’s institution has a sponsored research agreement with AstraZeneca. JAC has participated on an AstraZeneca advisory board. GGI was formerly employed by, and may hold stock in, Horizon Therapeutics. Support for the present study and manuscript editorial assistance were provided by AstraZeneca and Fishawack Health., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM. Limits of quantitation were determined by spiking synthetic peptides into a human spleen matrix. In addition, 20 clinical samples were analyzed using FAIMS-PRM and the quantitation of HER2 was compared with that obtained with the Ventana immunohistochemistry assay. FAIMS-PRM improved the overall signal-to-noise ratio over that from PRM and increased assay sensitivity in FFPE tissue analysis for four (HER2, EGFR, cMET, and KRAS) of five proteins of clinical interest. FAIMS-PRM enabled sensitive quantitation of basal HER2 expression in breast cancer samples classified as HER2 negative by immunohistochemistry. Furthermore, we determined the degree of FAIMS-dependent background reduction and showed that this correlated with an improved lower limit of quantitation with FAIMS. FAIMS-PRM is anticipated to benefit clinical trials in which multiple biomarker questions must be addressed and the availability of tumor biopsy samples is limited., (© 2022. The Author(s).)

Mass spectrometry-based targeted proteomics employs heavy isotope-labeled proteins or peptides as standards to improve accuracy and precision. The input sample amount is often determined by the total quantity of endogenous proteins or peptides, as defined by spectrophotometric assays, before the heavy-isotope standards are spiked into the samples. Errors in spectrophotometric measurements, which may be due to low sensitivity or chemical or biological interference, have a direct impact on the quantitative mass spectrometry results. Currently used targeted proteomics workflows cannot identify or correct deviations that arise from differences in the input sample amount. We have developed a workflow, global extraction from parallel reaction monitoring (PRM), to identify and quantify thousands of background peptides that are inherently acquired by PRM experiments. These background peptides were used to identify differences in the input sample amount and to reduce this variance by intensity-based, post-acquisition normalization. This approach was then applied to a xenograft study to improve the quantification of human proteins in the presence of mouse tissue contamination. In addition, these background peptides also provided a direct source of quality control metrics related to sample handling and preparation.

Plasmin is shown to specifically cleave vitronectin at the Arg361-Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-1 inhibitor of plasminogen activator (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI-1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.

Activation of blood platelets by thrombin was previously shown to specifically release protein kinase A, which in human plasma singles out and phosphorylates one protein, identified as vitronectin. This protein is known to be involved in processes that follow platelet stimulation, specifically, in the binding of heparin (interfering with the heparin-mediated inhibition of thrombin and Factor Xa by antithrombin III), in the growth of endothelial cells and in fibrinolysis. This paper shows that phosphorylation of vitronectin by protein kinase A is stoichiometric (approx. 1 mol/mol), that it is targeted to one site (Ser-378) at the C-terminal edge of the heparin-binding domain, and that it distinguishes between the two physiologically occurring forms of vitronectin: the one-chain (75 kDa) form, and the nicked two-chain (65 + 10 kDa) form, held together by an interchain disulphide bridge. Protein kinase A phosphorylates the one-chain form but not the two-chain form, although Ser-378 and the complete recognition sequence of the kinase are still present in the clipped 65 kDa chain. Cleavage of the Arg-379-Ala-380 bond results therefore in a conformationally distinct form of vitronectin in which Ser-378 is 'buried'. This is demonstrated by our finding that Ser-378 is present in the 65 kDa chain of clipped vitronectin but inaccessible to phosphorylation at physiological pH. Upon binding heparin, the phosphorylation site becomes exposed and able to undergo a stoichiometric phosphorylation at physiological pH.

In circulating blood, vitronectin occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Ser378 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Ser378 and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effectors involved in vitronectin function.

The catalytic subunit (C) of cAMP-dependent protein kinase selectively phosphorylates vitronectin, a plasma protein that promotes cell adhesion and platelet aggregation, inhibits the inactivation of thrombin by antithrombin III, and participates in complement function. This specific phosphorylation is used here (a) to develop an enzymatic assay for vitronectin (with C and [gamma-32P]ATP) which can be used to identify the vitronectin-containing fractions at each stage of its purification; (b) to radioactively label vitronectin and differentiate between the intact and the nicked form of this protein in structure-function studies; and (c) to identify possible vitronectin-related proteins in the plasma of other animal species.

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.