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Vaccination against COVID-19 has shown activation of different immune cell types. Here the authors characterise the immune response to the SARS-CoV-2 mRNA vaccine using longitudinal CyTOF single cell approaches to characterise antigen specific B and T-cell responses promoted by this vaccine.

Summary: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.

ABSTRACT Debate continues as to the role of combination antibiotic therapy for the management of Pseudomonas aeruginosa infections. We studied the extent of bacterial killing by and the emergence of resistance to meropenem and amikacin as monotherapies and as a combination therapy against susceptible and resistant P. aeruginosa isolates from bacteremic patients using the dynamic in vitro hollow-fiber infection model. Three P. aeruginosa isolates (meropenem MICs of 0.125, 0.25, and 64 mg/L) were used, simulating bacteremia with an initial inoculum of ~1 × 105 CFU/mL and the expected pharmacokinetics of meropenem and amikacin in critically ill patients. For isolates susceptible to amikacin and meropenem (isolates 1 and 2), the extent of bacterial killing was increased with the combination regimen compared with the killing by monotherapy of either antibiotic. Both the combination and meropenem monotherapy were able to sustain bacterial killing throughout the 7-day treatment course, whereas regrowth of bacteria occurred with amikacin monotherapy after 12 h. For the meropenem-resistant P. aeruginosa isolate (isolate 3), only the combination regimen demonstrated bacterial killing. Given that tailored antibiotic regimens can maximize potential synergy against some isolates, future studies should explore the benefit of combination therapy against resistant P. aeruginosa. IMPORTANCE Current guidelines recommend that aminoglycosides should be used in combination with β-lactam antibiotics as initial empirical therapy for serious infections, and otherwise, patients should receive β-lactam antibiotic monotherapy. Given the challenges associated with studying the clinical effect of different antibiotic strategies on patient outcomes, useful data for subsequent informed clinical testing can be obtained from in vitro models like the hollow-fiber infection model (HFIM). Based on the findings of our HFIM, we propose that the initial use of combination therapy with meropenem and amikacin provides some bacterial killing against carbapenem-resistant P. aeruginosa isolates. For susceptible isolates, combination therapy may only be of benefit in specific patient populations, such as critically ill or immunocompromised patients. Therefore, clinicians may want to consider using the combination therapy for the initial management and ceasing the aminoglycosides once antibiotic susceptibility results have been obtained.

Abstract Background The population pharmacokinetics of total and unbound posaconazole following intravenous administration has not yet been described for the critically ill patient population. The aim of this work was, therefore, to describe the total and unbound population pharmacokinetics of intravenous posaconazole in critically ill patients and identify optimal dosing regimens. Methods This was a prospective observational population pharmacokinetic study in critically ill adult patients with presumed/confirmed invasive fungal infection. A single dose of 300 mg posaconazole was administered intravenously as an add-on to standard antifungal therapy, and serial plasma samples were collected over 48 h. Total and unbound posaconazole concentrations, measured by chromatographic method, were used to develop a population pharmacokinetic model and perform dosing simulations in R using Pmetrics. Results From eight patients, 93 pairs of total and unbound concentrations were measured. A two-compartment linear model with capacity-limited plasma protein binding best described the concentration-time data. Albumin and body mass index (BMI) were included as covariates in the final model. Mean (SD) parameter estimates for the volume of the central compartment (V) and the elimination rate constant were 72 (43) L and 42.1 (23.7) h−1, respectively. Dosing simulations showed that high BMI was associated with a reduced probability of achieving target total and unbound posaconazole concentrations. Low serum albumin concentration was associated with a reduced probability of attaining target total but not unbound posaconazole concentrations. Conclusions An important clinical message of this study is that critically ill patients with increased BMI may require larger than approved loading doses of intravenous posaconazole when considering currently recommended dosing targets. Variability in plasma albumin concentration appears unlikely to affect dosing requirements when the assessment is based on unbound concentrations. Where available, therapeutic drug monitoring of unbound concentrations may be useful.

Microdialysis is a technique used to measure the unbound antibiotic concentration in the interstitial spaces, the target site of action. In vitro recovery studies are essential to calibrating the microdialysis system for in vivo studies. The effect of a combination of antibiotics on recovery into microdialysate requires investigation. In vitro microdialysis recovery studies were conducted on a combination of vancomycin and tobramycin, in a simulated in vivo model. Comparison was made between recoveries for three different concentrations and three different perfusate flow rates. The overall relative recovery for vancomycin was lower than that of tobramycin. For tobramycin, a concentration of 20μg/mL and flow rate of 1.0μL/min had the best recovery. A concentration of 5.0μg/mL and flow rate of 1.0μL/min yielded maximal recovery for vancomycin. Large molecular size and higher protein binding resulted in lower relative recoveries for vancomycin. Perfusate flow rates and drug concentrations affected the relative recovery when a combination of vancomycin and tobramycin was tested. Low perfusate flow rates were associated with higher recovery rates. For combination antibiotic measurement which includes agents that are highly protein bound, in vitro studies performed prior to in vivo studies may ensure the reliable measurement of unbound concentrations. Keywords: Microdialysis, Combination antibiotic therapy, Relative recovery rate, Pharmacokinetics, Anti-infectives, Protein binding

Over recent years, pigs have been promoted as potential animal model due to their anatomical and physiological similarities with humans. However, information about the contribution of distinct renal elimination processes [glomerular filtration rate (GFR), effective renal plasma flow (ERPF), tubular secretion, and reabsorption] in pigs is currently limited. Therefore, a cocktail of renal markers, consisting of iohexol (GFR), para-aminohippuric acid (ERPF and net tubular anion secretion), pindolol (net tubular cation secretion), and fluconazole (net tubular reabsorption) was administered intravenously to 7-week-old male conventional pigs. Plasma and urinary concentrations were determined using validated analytical methods. The clearance of iohexol (GFR) was 97.87 ± 16.05 ml/min/m² (mean ± SD). The ERPF, calculated as the renal clearance of PAH, was 226.77 ± 62.45 ml/min/m², whereas the net tubular secretion of PAH was 130.28 ± 52.62 ml/min/m². The net tubular secretion of R-pindolol and S-pindolol was 13.53 ± 12.97 and 18.01 ± 39.23 ml/min/m², respectively. The net tubular reabsorption of fluconazole was 78.32 ± 13.52 ml/min/m². Overall, this cocktail of renal markers was considered to be safe for use in pigs since no adverse effects were observed. Iohexol, PAH and fluconazole were considered suitable renal marker to assess the porcine renal function. Pindolol seems less appropriate due to the high degree of nonrenal clearance in pigs. The values of GFR, ERPF, and anion secretion are within the same range for both human and pig. Regarding the tubular reabsorption of fluconazole, slightly higher values were obtained for pigs. Nevertheless, these results indicate the conventional pig could be an appropriate animal model to study renal drug elimination processes in humans.

We evaluated piperacillin-tazobactam and tobramycin regimens against Pseudomonas aeruginosa isolates from critically ill patients. Static-concentration time-kill studies (SCTK) assessed piperacillin-tazobactam and tobramycin monotherapies and combinations against four isolates over 72 h. A 120 h-dynamic in vitro infection model (IVM) investigated isolates Pa1281 (MICpiperacillin 4 mg/L, MICtobramycin 0.5 mg/L) and CR380 (MICpiperacillin 32 mg/L, MICtobramycin 1 mg/L), simulating the pharmacokinetics of: (A) tobramycin 7 mg/kg q24 h (0.5 h-infusions, t1/2 = 3.1 h); (B) piperacillin 4 g q4 h (0.5 h-infusions, t1/2 = 1.5 h); (C) piperacillin 24 g/day, continuous infusion; A + B; A + C. Total and less-susceptible bacteria were determined. SCTK demonstrated synergy of the combination for all isolates. In the IVM, regimens A and B provided initial killing, followed by extensive regrowth by 72 h for both isolates. C provided >4 log10 CFU/mL killing, followed by regrowth close to initial inoculum by 96 h for Pa1281, and suppressed growth to 10 CFU/mL for CR380. A and A + B initially suppressed counts of both isolates to 10 CFU/mL, before regrowth to control or starting inoculum and resistance emergence by 72 h. Overall, the combination including intermittent piperacillin-tazobactam did not provide a benefit over tobramycin monotherapy. A + C, the combination regimen with continuous infusion of piperacillin-tazobactam, provided synergistic killing (counts 10 CFU/mL) of Pa1281 and CR380, and suppressed regrowth to 10 CFU/mL, respectively, and resistance emergence over 120 h. The shape of the concentration–time curve was important for synergy of the combination.

Abstract Background Nebulised antibiotics are frequently used for the prevention or treatment of ventilator-associated pneumonia. Many factors may influence pulmonary drug concentrations with inaccurate dosing schedules potentially leading to therapeutic failure and/or the emergence of antibiotic resistance. We describe a research pathway for studying the pharmacokinetics of a nebulised antibiotic during mechanical ventilation using in vitro methods and ovine models, using tobramycin as the study antibiotic. Methods In vitro studies using a laser diffractometer and a bacterial-viral filter were used to measure the effect of the type and size of tracheal tubes and antibiotic concentration on the particle size distribution of the tobramycin 400 mg (4 ml; 100 mg/ml) and 160 mg (4 ml, 40 mg/ml) aerosol and nebulised mass delivered. To compare the regional drug distribution in the lung of two routes (intravenous and nebulised) of drug administration of tobramycin 400 mg, technetium-99m-labelled tobramycin 400 mg with planar nuclear medicine imaging was used in a mechanically ventilated ovine model. To measure tobramycin concentrations by intravenous and nebulised tobramycin 400 mg (4 ml, 100 mg/ml) administration in the lung interstitial space (ISF) fluid and blood of mechanically ventilated sheep, the microdialysis technique was used over an 8-h duration. Results Tobramycin 100 mg/ml achieved a higher lung dose (121.3 mg) compared to 40 mg/ml (41.3 mg) solution. The imaging study with labelled tobramycin indicated that nebulised tobramycin distributed more extensively into each lung zone of the mechanically ventilated sheep than intravenous administration. A higher lung ISF peak concentration of tobramycin was observed with nebulised tobramycin (40.8 mg/l) compared to intravenous route (19.0 mg/l). Conclusions The research methods appear promising to describe lung pharmacokinetics for formulations intended for nebulisation during mechanical ventilation. These methods need further validation in an experimental pneumonia model to be able to contribute toward optimising dosing regimens to inform clinical trials and/or clinical use.

Resveratrol has been shown to preserve organ function and improve survival in hemorrhagic shock rat models. This study investigated whether seven days of oral resveratrol could improve hemodynamic response to hemorrhage and confer benefits on risk of acute kidney injury (AKI) without inducing coagulopathy in a canine model. Twelve greyhound dogs were randomly allocated to receive oral resveratrol (1000 mg/day) or placebo for seven days prior to inducing hemorrhage until a targeted mean blood pressure of ≤40 mmHg was achieved. AKI biomarkers and coagulation parameters were measured before, immediately following, and two hours after hemorrhage. Dogs were euthanized, and renal tissues were examined at the end of the experiment. All investigators were blinded to the treatment allocation. A linear mixed model was used to assess effect of resveratrol on AKI biomarkers and coagulation parameters while adjusting for volume of blood loss. A significant larger volume of blood loss was required to achieve the hypotension target in the resveratrol group compared to placebo group (median 64 vs. 55 mL/kg respectively, p = 0.041). Although histological evidence of AKI was evident in all dogs, the renal tubular injury scores were not significantly different between the two groups, neither were the AKI biomarkers. Baseline (pre-hemorrhage) maximum clot firmness on the Rotational Thromboelastometry (ROTEM®) was stronger in the resveratrol group than the placebo group (median 54 vs. 43 mm respectively, p = 0.009). In summary, seven days of oral resveratrol did not appear to induce increased bleeding risk and could improve greyhound dogs’ blood pressure tolerance to severe hemorrhage. Renal protective effect of resveratrol was, however, not observed.

Two peccary species, Mylohyus elmorei and Prosthennops serus are described from the medium-bodied fauna of the Gray Fossil Site (GFS) of northeastern Tennessee. This site, recognized as an oak-hickory forest, is latest Hemphillian or earliest Blancan based on mammalian biochronology, with an estimated age of 4.9–4.5 Ma. The GFS represents the only site outside the Palmetto Fauna of Florida with M. elmorei, greatly expanding the species range north over 920 km, well into the Appalachian region. This is also the first Appalachian occurrence of the relatively widespread P. serus. Our understanding of intraspecific variation for both M. elmorei and P. serus is expanded due to morphological and proportional differences found in cranial and dental material from the GFS, Tyner Farm locality, Palmetto Fauna, and within the literature. The GFS M. elmorei material represents the most complete mandible and second cranium for the species, and preserve intraspecific variation in the length of the diastema, dental proportions, and the complexity of the cuspules of the hypoconulid complex. Similarly, mandibular material from the GFS for P. serus exhibited larger dentitions and a greater degree of robustness than currently recognized for the species.

The wolverine (Gulo gulo) is the largest living terrestrial member of the Mustelidae; a versatile predator formerly distributed throughout boreal regions of North America and Eurasia. Though commonly recovered from Pleistocene sites across their range, pre-Pleistocene records of the genus are exceedingly rare. Here, we describe a new species of Gulo from the Gray Fossil Site in Tennessee. Based on biostratigraphy, a revised estimate of the age of the Gray Fossil Site is Early Pliocene, near the Hemphillian—Blancan transition, between 4.9 and 4.5 Ma. This represents the earliest known occurrence of a wolverine, more than one million years earlier than any other record. The new species of wolverine described here shares similarities with previously described species of Gulo, and with early fishers (Pekania). As the earliest records of both Gulo and Pekania are known from North America, this suggests the genus may have evolved in North America and dispersed to Eurasia later in the Pliocene. Both fauna and flora at the Gray Fossil Site are characteristic of warm/humid climates, which suggests wolverines may have become ‘cold-adapted’ relatively recently. Finally, detailed comparison indicates Plesiogulo, which has often been suggested to be ancestral to Gulo, is not likely closely related to gulonines, and instead may represent convergence on a similar niche.