The Etotal quantities corresponding to the best pairs: NPX-Eudragit L100, NaDLO–HPMC(P), DMF–HPMC(AS) and OPZ–HPMC(AS) were −143.38, −348.04, −110.42, and −269.43 kJ/mol, respectively. Using a HME experimental technique, few API–polymer pairs were successfully extruded. These extruded solid forms did not release APIs in a simulated gastric fluid (SGF) pH 1.2 environment but released them in a simulated intestinal fluid (SIF) pH 6.8 environment. The study demonstrates the compatibility between APIs and excipients, and finally suggests a potential polymeric excipient for each delayed-release API, which could facilitate the development of the solid dispersion of poorly soluble APIs for dissolution and bioavailability enhancement.Solid dispersion of poorly soluble APIs is known to be a promising strategy to improve dissolution and oral bioavailability. To facilitate the development and commercialization of a successful solid dispersion formulation, understanding of intermolecular interactions between APIs and polymeric carriers is essential. In this work, first, we assessed the molecular interactions between various delayed-release APIs and polymeric excipients using molecular dynamics (MD) simulations, and then we formulated API solid dispersions using a hot melt extrusion (HME) technique. To assess the potential API–polymer pairs, three quantities were evaluated: (a) interaction energy between API and polymer [electrostatic (E One of the most important challenges in the delivery of active pharmaceutical ingredients (APIs) faced by formulation scientists is how to counter the low aqueous solubility of APIs ,2,3. Amo®, Norvir®, FenoglideTM, Verapamil and Posaconazole.To develop the amorphous solid dispersion of APIs, a hot melt extrusion (HME) technique has emerged as a potent processing technology and several commercial HME products are available on the market or are under late-stage development ,7. In HMA major challenge for the development of amorphous solid dispersion formulations occurs due to the amorphous nature of APIs as they are thermodynamically unstable and tend to recrystallize . To mitiFor the successful development of a solid dispersion formulation, understanding of the intermolecular interactions between APIs and polymeric carriers is crucial ,21. In pIn addition to experimental explorations, molecular dynamics (MD) simulation has been widely employed as a promising tool to determine API-polymer miscibility in solid dispersions as well as formulation design ,30,31. FOur work involved using both MD simulation and experimental techniques to gain a better understanding of how delayed-release drug APIs interact with enteric polymeric excipients at the molecular level. This knowledge can help speed up the development of solid dispersions for delayed-release applications. It is worthwhile to note that the selected APIs are available in enteric-coated dosage form on the market; therefore, they are the ideal candidates for solid dispersion formulations. Precisely, first the compatibility between APIs and polymeric excipients was investigated by accessing molecular interactions using molecular dynamics (MD) simulations, which were later used to identify the potential API-polymeric pairs for solid dispersions. Then, solid dispersions of the suggested API-excipient pairs were prepared using HME to gauge the feasibility of the pairs, followed by release studies in both simulated gastric fluid (SGF) with a pH of 1.2 and simulated intestinal fluid (SIF) with a pH of 6.8.Polymer Builder module in Materials Studio , hydroxypropyl methylcellulose acetate succinate [HPMC(AS)], and Eudragit L100 were chosen. s Studio . These pructures . Using tructures ,38. The MD simulations were conducted in four separate sets. In the first set, the crystal structure of each drug API (NPX or NaDLO or DMF or OPZ) was simulated for 5 ns using isothermal and isobaric (NPT) MD simulation. This set of simulations was performed to verify the force field used herein. In the second set, a cubic simulation box (~6 nm each side) was built for each polymer excipient [HPMC(P) or HPMC(AS) or Eudragit L100]. Then, 10 API molecules of each drug were ramdomly added into the simulation boxes, resulting in 12 different simulation systems. The number of polymer chains was added in such a way that all systems maintained 5 wt% API and 95 wt% polymer excipients. For each system, 10 ns isothermal and isochoric (NVT) MD simulations were performed at 300 K, and the last 8 ns trajectory was used for analysis. A representative simulation snapshot for this type of system is shown in Experiments were conducted to assess the feasibility of mixing the API and polymeric excipient in a solid state once appropriate pairs of drug APIs and polymeric excipients were identified through MD simulations. The experimental procedures are elaborated later in this section. Afterwards, simulation systems were built for feasible API and excipient pairs in the third set, with a different API loading of 50 wt%. Similarly to the 2nd set, for this set, each system was simulated for 10 ns NVT MD simulations. The aim of this set was to analyze the effect of loading. In the final set, the simulations were performed at various temperatures.The GROMACS v5.1.2 package was used to conduct all simulations . The firFollowing the simulation, API polymer mixtures corresponding to 5 wt% and 50 wt% were prepared by accurately weighing powdered API and polymers in a screw-cap bottle and mixing them homogenously using Powder mixer  at 50 RPM for 30 min. NaDLO and OPZ were purchased from Jai Radhe Sales India. DMF and NPX base were supplied by Sigma-Aldrich, St. Louis, MO, USA. HPMC AS (LF grade) and HPMC-P (55 grade) was kindly provided by Shine Etsu Chemical Co. . Eudragit L100-55 was provided by Evonik Industries . High-performance liquid chromatography (HPLC) grade solvents were supplied by Fischer Scientific Pte. Ltd.,  and other reagents were supplied by Sigma, St. Louis, MO, USA and used as supplied. The solubilities, melting points, and degradation temperatures of the API and polymers are shown in For extruding solid dispersions in the form of cylindrical strands, a rod die with a 2 mm orifice was employed. The extruded strands were gathered on a conveyor belt, cooled with air, and then kept in screw-capped glass bottles under low humidity conditions (25% relative humidity) at room temperature. The resulting extrudates were subjected to ball milling using a stainless-steel vessel with a 1.5-cm diameter stainless-steel ball, at a frequency of 30 Hz for 2 min, using the MM 200 Retsch GmbH equipment from Haan, Germany. The milled extrudates were utilized for analyzing and characterizing the solid dispersions.−1.A Bruker D8 Advance powder X-ray diffractometer, which utilizes Cu Kα radiation (λ = 1.540 60 Å), an acceleration voltage of 35 kV, and a current of 40 mA power, was used to collect Powder X-ray diffraction (PXRD) data. Samples were scanned using a continuous scanning mode in the 2θ range from 5° to 50°, with a scan rate of 5° min−1 at a scan speed of 0.2 cm−1S−1 with a spectral resolution of 4 cm−1.A Frontier FT-IR/NIR Spectrometer  equipped with a mid-infrared (MIR) triglycine sulfate (TGS) detector was used to obtain FTIR transmission spectra of the APIs, physical mixtures, and extruded samples. A small quantity of powdered sample was secured on the sample holder and scanned over a range of 4000–650 cm(1)In Simulated Gastric fluid (SGF) pH 1.2 for 2 h: The samples were placed in a basket and dilution was tested for 2 h in SGF pH 1.2 with sample collections at 15, 30, 60, 90 and 120 min. A 2 mL sample was withdrawn after each time interval and fresh media were replenished. The samples were filtered through a 0.45 µm syringe filter and analyzed by HPLC.(2)In Simulated Intestinal fluid (SIF) pH 6.5 for 2 h: After 2 h in SGF, the dissolution medium was changed to SIF and the basket was lowered to run the same sample again. Samples were collected at 5, 10, 15, 30, 60, 90 and 120 min and 2 mL of fresh SIF was replenished after each collection. The samples were filtered through a 0.45 µm syringe filter and analyzed by HPLC.The extruded samples were broken as granules and passed through sieve no. 18 and retained in sieve no. 30 for dissolution studies. USP type 2 dissolution apparatus (Agilent Technologies) with baskets was employed for this study. All the samples were tested using the following sequence:For NaDLO solid dispersion dissolution, granules equivalent to 50 mg of NaDLO were used, whereas for DMF solid dispersion dissolution, granules equivalent to 120 mg DMF were used.To validate the force field of API molecules, first, the unit cell of each drug crystal was downloaded from the Cambridge Crystallographic Data Centre (CCDC) database. Then, the unit cell of each drug was extended in all directions to ensure that the dimensions in each direction were either close to or higher than 50 Å. To do so, NPX, NaDLO, DMF, and OPZ were extended to 4 × 9 × 7, 5 × 2 × 5, 14 × 10 × 7, and 5 × 5 × 5, respectively. After MD simulation, from the first set of simulations, densities and lattice parameters of all crystals were estimated and compared with the literature, as tabulated in coul), Lenard-Jones (ELJ), and total , between drug API molecules and polymer excipients, which were determined in the second set of simulations.To identify a suitable polymeric excipient for a stabilized amorphous API in solid dispersion formulation, solubility measurement is crucial. Typically, the solubility of a molecule in a specific medium can be gauged based on the interaction energy between interacting molecules; in general, the higher the interaction energy (stronger interaction), the higher the solubility ,46,47. Rtotal between NPX and polymeric excipient is dominated by van der Waals interactions (ELJ). Similarly to NPX, NaDLO–excipient interactions were negative  < HPMC(P). The Etotal between NaDLO and HPMC(P) was the highest, and thus is expected to be a potential choice. The possible reason for the highest interaction between NaDLO and HPMC(P) is the favourable interaction between hexagonal aromatic rings that are present in both structures  interactions due to the ionic nature of NaDLO.The interaction energies between NPX and polymeric excipients were negative and were found to increase in the order of HPMC(AS) < HPMC(P) < Eudragit L100 a. This inegative b. The Ecures see . Owing tcoul, ELJ, and Etotal increased in the order of Eudragit L100 < HPMC(P) < HPMC(AS), whereas for OPZ, these quantities increased in the order of HPMC(P) < Eudragit L100 < HPMC(AS). By carefully observing the chemical structures, one can say that, with a linear chain in Eudragit L100, a higher interaction with DMF  is expected. Interestingly, this was not observed here, instead HPMC(AS) showed the highest interaction with both DMF and OPZ. To unveil this behaviour, we further evaluated the intra-atomic Eudragit L100-Eudragit L100 and HPMC(AS)–HPMC(AS) interactions by radial distribution function g(r)asr is the distance between atoms i and j, iN and jN are the numbers of atoms i and j, j around i within a shell from r to r + Δr, respectively. g(r) of Eudragit L100 around Eudragit L100 and HPMC(AS) around HPMC(AS) in a mixture of these excipients with DMF and OPZ, based on all atoms. For DMF, as shown in r ~ 0.96 and 1.1 Å, indicating strong interaction between Eudragit L100 and Eudragit L100 as well as HPMC(AS) and HPMC(AS). However, Eudragit L100-Eudragit L100 interaction was stronger than HPMC(AS)-HPMC(AS), as reflected by a greater peak height in the former. Thus, owing to weaker intra-atomic HPMC(AS)–HPMC(AS) interaction, DMF is more accessible to HPMC(AS) compared to Eudragit L100 and thus has the highest interaction. Similarly, for OPZ  and HPMC(AS). Due to weaker intra-atomic HPMC(AS)-HPMC(AS) interaction, OPZ is more accessible to HPMC(AS) compared to Eudragit L100, and thus shows the highest interaction. In brief, the potential choice for both DMF and OPZ is HPMC(AS). Because both DMF and OPZ are neutral molecules, as is NPX, the Etotal between DMF/OPZ and the polymeric excipient is dominated by the van der Waals interaction rather than the electrostatic interaction.Similarly to NPX and NaDLO, DMF–excipient and OPZ–excipient interactions were negative c,d, indi for OPZ b, two prr) between a donor and an acceptor ≤ 3.5 Å and (2) the angle of hydrogen-donor-acceptor, α ≤ 30° 433 K < [DMF-HPMC(AS)]373 K < [DMF-HPMC(AS)]300 K. This indicates that DMF would be strongly bounded to HPMC(AS) at a lower temperature. As expected, energy ratios indicate the same hierarchy as the interaction trend  was considered. Particularly, simulations were performed at three different temperatures at 300 K, 373 K and 433 K, respectively. It should be noted that the examined temperatures are below the degradation temperature of DMF. on trend b. Furthecoul, ELJ, and Etotal increased in the order of HPMC(AS) < HPMC(P) < Eudragit L100, whereas for NaDLO, these quantities increased in the order of Eudragit L100 < HPMC(AS) < HPMC(P). For DMF and OPZ, the hierarchies of the energy terms are Eudragit L100 < HPMC(P) < HPMC(AS) and HPMC(P) < Eudragit L100 < HPMC(AS). The suggested API–polymer pairs are NPX-Eudragit L100, NaDLO-HPMC(P), DMF-HPMC(AS) and OPZ-HPMC(AS), respectively. The energy ratio trends of API–polymer combinations are consistent with the interaction energy hierarchies. All APIs were able to form hydrogen bonds with polymeric excipients. Being ionic in nature, NaDLO showed a higher number of hydrogen bonds compared to the other APIs. Among non-ionic APIs, OPZ had a higher number of hydrogen bonds. The pairs NaDLO–HPMC(P) and DMF–HPMC(AS) were successfully extruded by HME experiments. The release studies revealed that NaDLO and DMF APIs could be loaded up to 50 wt% in HPMC(P) and HPMC(AS), respectively, as these dispersions are rarely released in SGF but are mostly released in SIF. API–polymer interaction decreases as API loading increases, which leads to a higher mobility of API, thus boosting the release of API. The MSD indicates that with an increase in temperature, the mobility of API increases, resulting in a faster release of API. This study, in suggesting a potential polymeric excipient for delayed-release APIs, provides atomic-level insights into the compatibility between APIs and polymeric carriers, which could accelerate the rational design and development of a solid dispersion system for poorly soluble APIs.To investigate molecular interactions in solid dispersions, MD simulations and experimental approaches were adopted. The interaction energies between NPX and polymer in terms of E


Human parainfluenza viruses (PIV) are a leading cause of pediatric acute respiratory infection (ARI), yet limited data exist on PIV-associated hospitalizations, particularly from serotype PIV-4, a serotype reported to cause severe disease. We describe the characteristics and seasonality of PIV-associated ARI hospitalizations, focusing on PIV-4.During 12/01/2016-03/31/2020, we enrolled children hospitalized with fever or respiratory symptoms at the 7 children’s hospitals of the CDC’s New Vaccine Surveillance Network. Molecular testing for rhino/enterovirus (RV/EV), respiratory syncytial virus (RSV), influenza virus, PIV (serotypes 1-4), human metapneumovirus, and adenovirus were performed on mid-turbinate nasal or throat specimens collected from enrolled children. Parent interviews and chart abstractions were performed.A PIV was detected in 1,004 (6%) of 16,282 enrolled children. A PIV and another respiratory virus (most frequently with RV/EV or RSV) were detected in 336 (33%) children; 668 (67%) children tested positive for only 1 PIV (no co-detection of any other virus or co-detected PIV), including PIV-1 (33%), PIV-2 (15%), PIV-3 (40%), and PIV-4 (12%) (Table 1). Among children with only 1 PIV detected, children with PIV-4 infection were older (mean age 3.1 y) and more likely to have an underlying medical condition than children infected with other PIV serotypes. Discharge diagnoses of asthma, otitis media, and pneumonia were most common in children with PIV-4, and croup most common in those with PIV-1 & 2 (Table 2). PIV-1 had biennial fall seasonality; PIV-2 was detected mostly during the fall of 2018; PIV-3 detections peaked annually in the spring; and PIV-4 was detected year-round . One in 6 children required ICU care.PIV infections were detected in 6% of ARI hospitalizations and occurred throughout the year, with seasonal variation in serotype dominance. Half of children with PIV-related hospitalizations were healthy . Severe disease (requirement for ICU care) was common in all PIV-associated hospitalizations. Research on PIV vaccines is warranted, with consideration to include PIV-4.John V. Williams, MD, Merck: Grant/Research Support|Quidel: Board Member Marian G. Michaels, MD, MPH, Merck: Grant/Research Support|Viracor: Grant/Research Support Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Mary A. Staat, MD, MPH, CDC: Grant/Research Support|Cepheid: Grant/Research Support|Merck: Grant/Research Support|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Up-To-Date: Honoraria Elizabeth P. Schlaudecker, MD, MPH, Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support Geoffrey A. Weinberg, MD, Merck & Co: Honoraria


Invasive aspergillosis (IA) and invasive mucormycosis (IM) are life-threatening invasive fungal diseases (IFDs) that occur in critically ill and/or immunocompromised patients. Approved treatment options have significant limitations in pediatric patients. This study assessed the safety and outcomes of isavuconazonium sulfate (ISAV) for the treatment of IFD in this patient population.This phase 2, open-label, non-comparative study enrolled patients at 10 centers in Belgium, Spain, and the US between 2019 and 2022. Patients aged 1 to < 18 years with possible, probable or proven IA or IM per the 2008 (EORTC/MSG) criteria received ISAV 10 mg/kg (max. 372 mg) every 8 h on days 1 and 2, and once-daily thereafter , for ≤ 84 days (IA) or ≤ 180 days (IM). Primary objectives were safety, including treatment-emergent adverse events (TEAEs), drug-related TEAEs, vital signs, electrocardiograms and laboratory parameters. Other key outcomes included all-cause case fatality through Day 42, overall response as assessed by an independent committee, and plasma ISAV levels. Data were summarized descriptively.Overall, 31 patients aged 1–17 years were enrolled; 80.6% were female and 61.3% were White (Table 1). The most common primary underlying condition was hematologic malignancy (58.1%). Patients received ISAV for a median (range) duration of 55 (2–181) days. Plasma ISAV levels were consistent with those seen in adults receiving the standard dose (Table 2). TEAEs occurred in 29 (93.5%) patients for a total of 415 events. Nine (29.0%) patients experienced drug-related TEAEs, and treatment was withdrawn in 2 patients due to TEAEs. Serious TEAEs occurred in 18 (58.1%) patients and were assessed as drug-related by the investigator in 1 patient (3.2%). All-cause case fatality through Day 42 was 6.5%. Overall, successful response rates were 54.8% at the end of treatment .ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia (includes relapsed disease); B-LL, B-cell lymphoblastic leukemia/lymphoma; EORTC/MSG, European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group; FAS, full analysis set; IA, invasive aspergillosis; IFD, invasive fungal disease; IM, invasive mucormycosis; MDS to AML, myelodysplastic syndrome transformed to AML; NHL, non-Hodgkin lymphoma; SD, standard deviation. An investigator assessment of IFD diagnosis was used. The FAS included all patients who are enrolled and receive at least one dose of study drug. Patients with possible IFD were eligible for enrollment; diagnostic tests to confirm the disease as ‘probable’ or ‘proven’ according to EORTC/MSG 2008 criteria were completed within 10 days after first dose of study drug. Other IFDs were defined as IFDs confirmed not to be IA or IM.EORTC/MSG, European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the Mycoses Study Group; IA, invasive aspergillosis; IFD: invasive fungal disease; IM, invasive mucormycosis; IV, intravenous; PKAS, pharmacokinetic analysis set; SAF, safety analysis set; SD, standard deviation. An investigator assessment of IFD diagnosis was used. Possible IFD was defined according to EORTC/MSG 2008 mycological criteria. Other IFDs were defined as IFDs confirmed not to be either IA or IM. The FAS included all patients who are enrolled and receive at least one dose of study drug. The PKAS consisted of all patients who took at least 1 dose of study drug and recorded least 1 plasma concentration measurement.AC, adjudication committee; FAS, full analysis set; IA, invasive aspergillosis; IFD: invasive fungal disease; IM, invasive mucormycosis; EOT, end of treatment; SAF, safety analysis set; TEAE, treatment-emergent adverse event. An investigator assessment of IFD diagnosis was used. Possible IFD was defined according to EORTC/MSG 2008 mycological criteria. Other IFDs were defined as IFDs confirmed not to be either IA or IM. If patient did not reach Day 42 or Day 84 of therapy, then the AC did not perform these assessments. The frequency 'N' in a column heading represents the number of patients included in the investigator assessment of IFD diagnosis. Overall response was based on a composite of clinical, mycological, and radiological responses with success criteria assessed. Overall response was considered 'Not evaluable' when one of the composite responses was not assessed. TEAEs were defined as adverse events observed after starting the study drug through 30 days after the last dose. Drug-related events, as assessed by the investigator, were those with a reasonable possibility that the event may have been caused by the study drug; if any other relationship was missing, the event was also considered as drug-related. The FAS and SAF were equivalent, and included all patients who were enrolled and received at least one dose of study drug.Treatment with ISAV in 31 immunocompromised pediatric patients with IFDs was well tolerated; only one (3.2%) patient had a serious drug-related TEAE. Plasma drug levels were also similar to those in adult patients. Overall favorable response was observed in > 50% of patients at EOT. All-cause case fatality rates were low (6.5%) at Day 42.Antonio C. Arrieta, MD, FIDSA, FPIDS, Astellas Pharma Global Development, Inc.: Advisor/Consultant|Astellas Pharma Global Development, Inc.: Grant/Research Support|Astellas Pharma Global Development, Inc.: Honoraria|Cumberland Pharmaceutical: Grant/Research Support|IDbyDNA: Advisor/Consultant|IDbyDNA: Grant/Research Support|Melinta: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Nabriva: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support|Pfizer, Inc: Advisor/Consultant|Pfizer, Inc: Grant/Research Support|Roche/Genentech: Grant/Research Support|The Medicine Company: Grant/Research Support Heidi Segers, MD, PhD, ALLTogether Consortium: Board Member|Astellas Pharma Global Development, Inc.: Support for the present publication|Jazz Pharmaceuticals: Support for attending meetings and/or travel|Stand up against Cancer Grant /C1 KU Leuven Grant: Grant/Research Support Jaime G. Deville, MD, Astellas Pharma Global Development, Inc.: Support for the present publication William J. Muller, MD, PhD, Adagio Therapeutics: Advisor/Consultant|Ansun Biopharma: Grant/Research Support|Astellas Pharma Global Development, Inc.: Grant/Research Support|Astellas Pharma Global Development, Inc.: Support for the present publication|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|DiaSorin Molecular LLC: Advisor/Consultant|Eli Lilly and Company: Grant/Research Support|Enanta Pharmaceuticals: Grant/Research Support|F. Hoffmann-La Roche: Grant/Research Support|Finley Law Firm, P.C: Expert Testimony|Gilead Sciences: Grant/Research Support|Janssen Biotech: Grant/Research Support|Karius, Inc.: Grant/Research Support|Melinta Therapeutics, Inc.: Grant/Research Support|Merck: Grant/Research Support|Moderna: Grant/Research Support|Nabriva Therapeutics, plc: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|Pfizer: Grant/Research Support|Sanofi Pasteur LLC: Advisor/Consultant|Tetraphase Pharmaceuticals, Inc.: Grant/Research Support Angela Manzanares, MD, Astellas Pharma Global Development, Inc.: Support for the present publication|Gilead Sciences: Support for attending meetings and/or travel Michael N. Neely, MD, Astellas Pharma Global Development, Inc.: Advisor/Consultant|Astellas Pharma Global Development, Inc.: Support for the present publication Victoria Bordon, MD, PhD, Astellas Pharma Global Development, Inc.: Support for the present publication Benjamin Hanisch, MD, American Society of Transplantation: Board Member|Astellas Pharma Global Development, Inc.: Support for the present publication|NIH: Site PI for International Pediatric Fungal Network- funds to institution Alvaro Lassaletta, MD, PhD, Astellas Pharma Global Development, Inc.: Support for the present publication|Gilead Sciences: Stocks/Bonds Brian T. Fisher, DO, MPH/MSCE, Allovir: Grant/Research Support|Astellas Pharma Global Development, Inc.: Support for the present publication|Merck: Grant/Research Support|Pfizer: Grant/Research Support Julie Autmizguine, MD, MHS, Astellas Pharma Global Development, Inc.: Advisor/Consultant|Astellas Pharma Global Development, Inc.: Support for the present publication Andreas H. Groll, MD, Amplyx: Advisor/Consultant|Astellas: Advisor/Consultant|Astellas: sered at the speakers’ bureau|Basilea: Advisor/Consultant|Basilea: served at the speakers’ bureau|F2G: Advisor/Consultant|F2G: served at the speakers’ bureau|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: served at the speakers’ bureau|Merck Sharp & Dohme LLC: Advisor/Consultant|Merck Sharp & Dohme LLC: Grant/Research Support|Merck Sharp & Dohme LLC: served at the speakers’ bureau|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: served at the speakers’ bureau Shamim Sinnar, MD, PhD, Astellas Pharma Global Development, Inc.: Astellas Employee Rodney Croos-Dabrera, PhD, Astellas Pharma Global Development, Inc.: Astellas Employee Marc Engelhardt, MD, Astellas Pharma Global Development, Inc.: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Mark E. Jones, PhD, Astellas Pharma Global Development, Inc: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Laura Kovanda, PhD, Astellas Pharma Global Development Inc.: Astellas Employee


Prolonged SARS-CoV-2 infections in immunocompromised hosts may predict or source the emergence of highly mutated variants of concern. The types of immunosuppression placing patients at highest risk for prolonged infection and associated intra-host viral evolution remain unclear.Adults aged ≥18 years were enrolled and followed at 5 hospitals in the Investigating Respiratory Viruses in the Acutely Ill (IVY) Network from 4/11/2022 – 2/1/2023. Eligible patients were SARS-CoV-2-positive by RT-qPCR in the previous 14 days and had an immunocompromising condition, including malignancy, solid organ or hematopoietic stem cell transplant (SOT/HSCT), autoimmune/autoinflammatory condition on immunosuppression, AIDS, or primary immunodeficiency. Nasal specimens were collected and tested by RT-qPCR every 2–4 weeks until negative in 2 consecutive specimens. All specimens underwent viral culture and whole genome sequencing. A Cox proportional hazards model was used to assess factors associated with prolonged infection.150 patients were enrolled with the following conditions: B cell malignancy or anti-B cell therapy (n=18), SOT/HSCT (n=59), AIDS (n=5), non-B cell malignancy (n=23), and autoimmune/autoinflammatory . 37 (25%) were RT-qPCR-positive and 11 (7%) were culture-positive ≥21 days after infection onset. Patients with B cell dysfunction had prolonged infection compared to those with autoimmune/autoinflammatory conditions  . The within-host evolutionary rate was similar in prolonged (≥ 21 days) and shorter (< 21 days) infections . Consensus spike mutations were identified in 4 individuals who were RT-qPCR-positive ≥ 56 days; 68% were in the receptor-binding domain (RBD) . The common spike mutations in this analysis were rare (< 2%) in global circulation.In this prospective cohort of immunocompromised patients during the Omicron variant period, prolonged SARS-CoV-2 infections were uncommon. While the within-host evolutionary rates are similar between prolonged and shorter infections, individuals with infections lasting ≥ 56 days accumulated mutations in the spike protein. These appear distinct from those seen globally.Bisected squares indicate more than one genomic mutation produced the same amino acid mutation. RBD is highlighted in gray.Emily T. Martin, PhD, MPH, Merck: Grant/Research Support Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support Carlos G. Grijalva, MD, MPH, AHRQ: Grant/Research Support|CDC: Grant/Research Support|FDA: Grant/Research Support|Merck: Advisor/Consultant|NIH: Grant/Research Support|Syneos Health: Grant/Research Support Adam S. Lauring, MD, PhD, Roche: Advisor/Consultant|Sanofi: Advisor/Consultant


Prediction of fluid responsiveness in acutely ill patients might be influenced by a number of clinical and technical factors. We aim to identify variables potentially modifying the operative performance of fluid responsiveness predictors commonly used in clinical practice.A sensitive strategy was conducted in the Medline and Embase databases to search for prospective studies assessing the operative performance of pulse pressure variation, stroke volume variation, passive leg raising (PLR), end-expiratory occlusion test (EEOT), mini-fluid challenge, and tidal volume challenge to predict fluid responsiveness in critically ill and acutely ill surgical patients published between January 1999 and February 2023. Adjusted diagnostic odds ratios (DORs) were calculated by subgroup analyses (inverse variance method) and meta-regression (test of moderators). Variables potentially modifying the operative performance of such predictor tests were classified as technical and clinical.p < 0.05 for all adjusted vs. unadjusted DORs). In addition, the operative performance of fluid responsiveness predictors was also influenced by clinical variables such as the positive end-expiratory pressure (in the case of EEOT) and the dose of norepinephrine used during the fluid responsiveness assessment for PLR and EEOT .A total of 149 studies were included in the analysis. The volume used during fluid loading, the method used to assess variations in macrovascular flow  in response to PLR/EEOT, and the apneic time selected during the EEOT were identified as technical variables modifying the operative performance of such fluid responsiveness predictor tests (Prediction of fluid responsiveness in critically and acutely ill patients is strongly influenced by a number of technical and clinical aspects. Such factors should be considered for individual intervention decisions.The online version contains supplementary material available at 10.1186/s13054-023-04629-w. Fluid therapy is one of the first-line interventions used to reverse tissue hypoperfusion during acute circulatory failure. In general, fluid administration aims to increase cardiac output (CO), supposing that this will improve tissue perfusion and then cellular respiration and cell metabolism . NeverthA number of maneuvers have been described to assess fluid responsiveness in acutely and critically ill patients . These iThe performance of predictor tests to assess fluid responsiveness might be affected by a number of clinical and technical factors , which chttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021266950) in August 2021.This meta-analysis was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) for diagnostic test accuracy (DTA) guidelines . The comA comprehensive search strategy was conducted using the Medline and Embase databases between January 1999 and February 2023. Data extraction and eligibility assessment were standardized and independently performed by two reviewers (J.I.A.S. and J.D.C.R.). Reference lists of selected manuscripts were also manually reviewed to search for potential studies not retrieved by the original search. No language restriction was applied. The complete search strategy and the terms used are available in the protocol uploaded to PROSPERO (registration number CRD42021266950).P-Patients and setting: We selected studies including critically ill and acutely ill perioperative patients. We excluded studies conducted in the emergency room and those including patients < 18 years of age and pregnant women.I-index test: We selected studies evaluating the operative performance of PPV, SVV, PLR, EEOT, MFC, and VTC as predictors of fluid responsiveness. Studies evaluating PPV and SVV performance were selected when they included patients under mechanical ventilation without respiratory effort and arrhythmias. Meanwhile, studies evaluating PLR-induced changes in both cardiac output and pulse pressure, including patients with intra-abdominal hypertension (explicit definition), were not selected.C-comparison or reference standard: Only studies including an explicit definition of fluid responsiveness (reference standard) after fluid loading or another maneuver (such as PLR or EEOT) were incorporated into the analysis.O-outcomes or target condition: We selected only studies reporting data about the operative performance of any fluid responsiveness test. Some studies had several data points on operative performance; in such cases, all data regarding operative performance were included.S-studies: We selected prospective studies. Meanwhile, case reports, studies with incomplete data, studies conducted in animals, studies assessing patients with heart failure, those including patients under one-lung ventilation or open chest surgery, and retrospective studies were excluded from the analysis.Studies were selected according to the PICOS statement as follows:Two authors (J.I.A.S. and J.D.C.R.) independently reviewed the titles and potentially eligible abstracts. Studies fulfilling the inclusion criteria were pooled in individual extraction sheets to be ultimately compared. Discrepancies about inclusion, quality, adequacy of data, and final classification of eligible studies were resolved by consensus among the authors.Data extracted from eligible studies included year of publication, authors, number of participants, sensitivity, specificity, area under the curve (AUC); method employed to assess preload dependency : fluid challenge, PLR, EEOT and Trendelenburg; technique used to assess the selected excitation method; variable employed for appraising fluid responsiveness , and clinical setting. Variables potentially modifying the operative performance of predictors of fluid responsiveness were classified as technical and clinical. Technical variables included fluid loading > or ≤ 4 mL/kg; hemodynamic variables were used to determine the PLR/EEOT response, which were separated into two groups: (a) CO direct measurements: aortic blood flow (ABF), CO/cardiac index (CI), stroke volume (SV)/stroke volume index (SVI), and velocity–time integral (VTI); and (b) CO surrogates: pulse pressure (PP), SVV and PPV. Other technical variables included the time lapse of expiration during the EEOT maneuver ; the threshold used to define fluid responsiveness (< or ≥ 10%); the use or not of thermodilution as a gold standard to measure CO; and whether the volume of MFC was considered a part of the fluid load during the FR assessment. Meanwhile, clinical variables included the type of patient, driving pressure, level of positive end-expiratory pressure (PEEP), tidal volume (Vt), total respiratory compliance, supine vs. prone position ventilation, and norepinephrine dose (< or ≥ 0.3 mcg/kg/min).The quality of eligible studies was assessed according to the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) using 4 domains: patient selection, index test, reference standard, and flow and time. Each area was evaluated for risk of bias and classified as low, high, or unclear. Moreover, the risk of applicability was considered in three domains: patient selection, index test, and reference standard, which were in turn assessed as low, high, or unclear .We computed the sensitivity, specificity, and diagnostic odds ratio (DOR) using a contingency table. In certain studies, operative performance was assessed in multiple subgroups, including different ventilation parameters, techniques used to assess fluid responsiveness, cutoff values to consider the test as positive, and methods for evaluating fluid response. In such cases, all the available data related to the operative performance were incorporated.DORs were calculated as described elsewhere (21). Higher DORs identify predictors with higher sensitivity and specificity combined with lower rates of false positives and negatives.I2).Fitted data of sensitivity, specificity, and AUC were assessed by using bivariate and hierarchical analyses. The summary receiver operating characteristic (ROC) curve was assessed by the Rutter and Gatsonis method, while the AUC was graduated according to Fisher et al. . UnadjusAsymmetry was assessed by Egger tests . PublicaAdjusted DORs were calculated by subgroup analyses (inverse variance method) and meta-regression (test of moderators) for all technical and clinical variables potentially influencing the operative performance of predictors of fluid responsiveness, as described above. Meta-regression was especially used to assess continuous variables such as Vt, lung compliance, PEEP, and driving pressure.A meta-regression was performed based on the quality assessment of all studies included (according to the QUADAS-2).The sensitivity analysis is summarized in Table P < 0.05 was considered statistically significant.The Grading of Recommendations Assessment, Development, and Evaluations (GRADE) was performed as a diagnostic test . The thrFrom 857 preliminary records identified from the EMBASE and Medline databases and their reference lists and after removing duplicated records and studies not fulfilling the inclusion criteria, a total of 149 studies were finally included in the analysis .The risk of bias is depicted in Fig. p = 0.01), and the studies that evaluated MFC and VTC were homogeneous (p > 0.05).In general, heterogeneity was moderate for PPV, SVV, PLR, and EEOT  , passive leg raising (PLR), and the end-expiratory occlusion test (EEOT). When fluid loading exceeded 4 ml/kg, the predictive performance of such fluid responsiveness predictors increased, as evidenced by the increase in the diagnostic odds ratio (DOR). Conversely, when fluid doses were ≤ 4 ml/kg, the opposite trend was observed, resulting in a decrease in the DOR values for all three predictors (5) Table .p < 0.01), while its predictive performance significantly decreased when the time lapse exceeded 30 s (p < 0.01) (Table The hemodynamic variables used to determine the PLR/EEOT response were found to significantly influence their predictive performance. Direct macro-flow measurements such as aortic blood flow (ABF), cardiac output (CO) or cardiac index (CI), stroke volume (SV) or stroke volume index (SVI), and velocity–time integral (VTI) increased its predictive performance Table . Convers1) Table .The operative performance related to other variables evaluated is shown in the Additional file p < 0.01)  values improved the ability of the end-expiratory occlusion test (EEOT) to predict fluid responsiveness. In fact, increasing PEEP by just 1.21 cmH2O led to a significant improvement in EEOT performance (1) Table .p > 0.05) (Table Higher doses of norepinephrine (≥ 0.3 mcg/kg/min) were related to significant improvements in the performance of PLR and EEOT to predict fluid responsiveness, while lower doses had the opposite effect , as evaluated by both the rho correlation coefficient and the Moses–Shapiro–Littenberg test. The accuracy of PPV as a diagnostic measure was deemed moderately reliable because the results from the studies included lack precision and might not be applicable to all clinical settings. Conversely, the accuracy of other predictors was deemed highly reliable. Additional information on the GRADE assessment can be found in Appendix Table S12-Table S17 of the Additional file There was no threshold effect for any predictor (p > 0.05)  , SVV, and PPV led to a decrease in operative performance. This phenomenon may be due to the relationship between CO surrogates and other physiological variables , such asThe duration of the respiratory hold constitutes a critical technical variable that demands careful consideration during the execution of this test. Notably, the operative performance displays an augmentation when the EEOT duration is ≤ 15 s, while a decline is observed when it extends to ≥ 30 s. This trend might be attributed to the fact that the maximal alteration in venous return and cardiac output typically materializes within the initial 15 s, with subsequent reductions occurring due to the finite nature of this change . MoreoveAlthough some authors have suggested that including the volume of the MFC within the fluid loading might change its operative performance , our metOur findings reported a positive relationship between EEOT operative performance and PEEP, which could be explained for two reasons. First, increasing PEEP could improve pulmonary compliance  due to an increase in cyclic changes in intrathoracic pressure induced by EEOT, thus increasing its operative performance . Second,Our data also suggest that doses of norepinephrine > 0.3 mcg/kg/min result in significant increases in the operative performance of PLR and EEOT. Previous studies have described that using norepinephrine is associated with fluid responsiveness . The likOur meta-analysis has several limitations. First, while the diagnostic odds ratio (DOR) summarizes the relationship between sensitivity and specificity, it does not indicate the direction of the relationship (which may vary among different predictors), even at similar DOR values . Second,Based on our findings, we recommend conducting a PLR/EEOT test with direct CO measurement. For the EEOT, the respiratory hold should be limited to less than 15 s. Physicians should be aware that higher positive end-expiratory pressure (PEEP) levels enhance the performance of EEOT. Additionally, the use of norepinephrine has been observed to enhance the operative performance of external fluid loading maneuvers, such as PLR and EEOT. Finally, when performing a fluid challenge, a volume of less than 4 ml/kg is suggested.Additional file 1. Supplement Material Appendix.


We read the meta-analysis by Messina et al.  on the eThis meta-analysis included studies involving patients with an open chest, ventilated with a low tidal volume, or undergoing laparoscopic surgery . In these conditions, PPV and SVV are known to be unreliable to predict fluid responsiveness . IncludiWe appreciate the fact that the predictive value of PPV and SVV was assessed in various subgroups. Regrettably, it was not evaluated in the subgroup of patients meeting all the conditions conducive to the reliable use of PPV and SVV. What was the area under the curve (AUC) in the subgroup of patients undergoing non-laparoscopic surgery with a closed chest and a tidal volume of 7–9 ml/kg? Would it support the conclusion that PPV and SVV are only moderately accurate to predict fluid responsiveness?We firmly believe that both PPV and SVV serve as reliable predictors of fluid responsiveness, provided that physiologic limitations to their use are respected , 4. SincFinally, unlike SVV monitoring, PPV monitoring does not require any cardiac output monitoring device. In a meta-analysis assessing the respective performance of PPV and SVV, it would have been wise to highlight this practical advantage as well.In summary, physiologic limitations to the use of PPV and SVV should be respected not only in clinical practice but also in meta-analyses; otherwise, they may lead to misleading conclusions. When these limitations are respected, we believe that both PPV and SVV are valuable variables for predicting fluid responsiveness and personalizing hemodynamic management, potentially leading to improved patient outcomes .


Fracturing fluids are widely applied in the hydraulic fracturing of shale gas reservoirs, but the fracturing fluid flowback efficiency is typically less than 50%, severely limiting the shale gas recovery. Additionally, the mechanism and main influencing factors of fracturing fluid flowback are unclear. In this study, microscopic experiments are conducted to simulate the fracturing fluid flowback progress in shale gas reservoirs. The mechanism and factors affecting fracturing fluid flowback/retention in the fracture zone were analyzed and clarified. Results show that the ultimate flowback efficiency of fracturing fluid is positively correlated with the fracturing fluid concentration and the gas driving pressure difference. There are four kinds of mechanisms responsible for fracturing fluid retention in the pore network: viscous resistance, the Jamin effect, the gas blockage effect and the dead end of the pore. Additionally, the ultimate flowback efficiency of the fracturing fluid increases linearly with increasing capillary number. These insights will advance the fundamental understanding of fracturing fluid flowback in shale gas reservoirs and provide useful guidance for shale gas reservoirs development. Nowadayrecovery ,7. TherePolymers are frequently employed in water-based fracturing fluids with the primary purposes of minimizing turbulence-induced friction and enhancing the fracturing fluid’s ability to carry proppant particles. This is achieved through the elevation of the fracturing fluid’s viscosity, transforming it into a non-Newtonian fluid . The comBased on the flow mechanism in the shale reservoir after hydraulic fracturing, scholars have applied different models to simulate the flow of fluids during the flowback process. From the early single-phase flow , to two-When gravity is negligible, the capillary force is an important factor influencing the transport of immiscible two-phase fluids at the pore level . The capIn order to investigate the factors influencing the fracture fluid flowback efficiency in shale gas reservoirs, microscopic models are applied in this study. In this field of application, a microscopic model is fabricated with a custom designed porous network etched on its surface that is used to inject desired solutions at certain operating conditions such as invasion amounts, flowback rates and injection pressures . ExperimA new polyacrylamide polymer fracturing fluid provided by Dingshan gas field was used in this study. PAM with a molecular weight of 26.8 million Daltons, a solid content of 89.0% and a hydrolysis degree of 19.7%. From a molecular design perspective, incorporating micro-block molecule models in polymerization maintains a linear molecular structure and improves solubility. The charge in the middle of each block is minimally affected by the environment, maintaining a certain degree of Coulomb repulsion between them, which results in a relatively large hydrodynamic radius for the polymer, thus maintaining high molecular weight and solution viscosity. The molecular structure characteristics are shown in −1, and the temperature was 20 °C). High-purity nitrogen with a concentration of 99.999% and a viscosity of 0.01757 mPa·s  was used as the experimental gas. The interfacial tension (IFT) between nitrogen and fracturing fluid was measured by a tensiometer at 20 °C. The contact angles of fracturing fluid-nitrogen and glass surface were measured using KRUSS DSA100S , as shown in Experimental fracturing fluids with concentrations of 0.1 wt%, 0.8 wt% and 1.2 wt% were prepared using DI water, with viscosities of 7.1 mPa·s, 62.7 mPa·s and 96.1 mPa·s, respectively , then observe and record the retention and distribution of the liquid phase in the microscopic model at each pressure, and close the nitrogen cylinder when there was no longer any significant change;Clean the model and repeat steps 1–3 after changing the fracturing fluid concentration and adjusting the nitrogen injection pressure.The specific experimental procedure was as follows:All experiments are conducted on the identical microscopic model, with every individual experiment preceded by a cleaning sequence that includes multiple rounds of DI water flushing, vacuuming, heating, and drying. Then, place the model under the microscope to ensure that there is no residual liquid in the pore network.FB%) can be calculated as shown in Equation (1):In order to quantify the fracturing fluid flowback and to further compare the flowback efficiencies of the different experimental groups, the experimental images were processed using ImageJ software. The experimental image was first sharpened, then the background was removed and the image was converted to a grey-scale binary image. In the binary image, the fracturing fluid is highlighted by the difference in gray-scale values between the fracturing fluid and the nitrogen ,26. The The results of the fracturing fluid flowback experiments are shown in The process of fracturing fluid flowback from the branch fractures to the main fracture was shown in To assess the impact of fracturing fluid concentration on the fluid flowback behavior, the experimental procedure was conducted once more with a heightened fracturing fluid concentration of 0.8 wt%. Additionally, the gas driving pressure differences remained at 0.1, 0.2 and 0.3 MPa for the three groups. The process of fracturing fluid flowback was shown in Compared to the first two sets of experiments, the third set further increased the fracturing fluid concentration (1.2 wt%), while adjusting the gas driving pressure differences to 0.3, 0.4 and 0.5 MPa. As shown in Viscous retention occurs when the fracturing fluid adheres to the solid phase surface due to the force of viscosity, as shown in As the pore radius changes, the fracturing fluid is not easily displaced at the smaller pore throat due to the Jamin effect , as shoThe gas blockage effect  causes the fracturing fluid to be trapped and unable to flow, thus resulting in the fracturing fluid being retained at the pore wall, as shown in The fracturing fluid becomes confined in the dead end, as shown in Based on the findings of the image analysis, it has been determined that there exist four types of fracturing fluid retention within the microscopic model:The main causes of the fracturing fluid retention described above are two types of resistance to the fracturing fluid flowback process: viscous force and capillary force.The main mechanism of viscous retention, shown in Viscosity is a special property of fluids. In a flowing fluid, if the layers are flowing at different velocities, there will be a pair of forces acting and reacting to slow down the original faster layer of flow and speed up the slower one. This pair of equal and opposite forces impede the relative motion of the flow layer. This property of the fluid is called viscosity, and this pair of forces is the viscous force. Viscous retention is an important mechanism of fracturing fluid retention in gas-fracturing fluid two-phase systems. As a polymer solution, PAM fracturing fluid has a reticulated structure. When flowing through porous media at low velocity, the shear effect is weak and the intermolecular forces between polymer molecules are strong, making it difficult to destroy the reticulated structure of the polymer solution ,29. As ar is the capillary radius, mm; dP is the driving pressure difference, Pa; σ is the gas-fracturing fluid interfacial tension, mN/m; θ is the three-phase contact angle of glass/fracturing fluid/gas, °; μ is the fracturing fluid viscosity, mPa·s; L is the length of fracturing fluid infiltration, mm.According to the Poiseuille formula, the volume of fracturing fluid driven out of the capillary isAccording to Equation (2), viscous retention is mainly controlled by the combination of fracture width, fracture length, driving pressure difference and fracturing fluid viscosity.As the concentration of the fracturing fluid increases, the microstructure of the polymer solution changes. Experiments were carried out using a HITACHI Regulus 8100 cryogenic scanning electron microscope (cryo-SEM) to observe the microstructure of the fracturing fluid, as shown in The main component of the fracturing fluid is the polymer hydrogel, where the polymer molecules are disordered in the solution, forming a coarse and uneven network structure. As concentration rises, the reticulated structure densifies, heightening intermolecular forces and rendering the structure more resistant to be destroyed during flowback, thereby considerably enhancing fracturing fluid retention. As shown in σ is the gas-fracturing fluid interfacial tension, mN/m; θ is the three-phase contact angle of glass/fracturing fluid/gas, °; r is the capillary radius, mm.The main mechanism of fracturing fluid retention caused by the Jamin effect, the gas blockage effect and the dead end of the pore, shown in During the gas drive process, the fracturing fluid in the dominant channel is driven out first, with the residual fracturing fluid remaining mainly as a “thin film” on the wall due to low capillary forces. Meanwhile, in the secondary fracture and pore, the residual fracturing fluid remains as a “thick film” with high saturation. Capillary force is the major factor in the retention of residual fracturing fluid both on the wall and in the pore. From Equation (3) it can be seen that the capillary force gradually increases as the pore radius decreases. Shale gas reservoirs contain a large fraction of nano pores . Consequ(1)The ultimate fracturing fluid flowback efficiencies decreased as the fracturing fluid concentration increased;(2)The ultimate fracturing fluid flowback efficiencies increased as the gas driving pressure differences increased, and this phenomenon was more obvious in the high concentration fracturing fluid.For the three fracturing fluid concentrations, a total of nine groups of experiments, as shown in v is the average flow velocity of the fluids in the microscopic model, m/s; μ is the nitrogen viscosity, mPa·s; σ is the gas-fracturing fluid interfacial tension, mN/m; θ is the three-phase contact angle of glass/fracturing fluid/gas, °.The fracturing fluid flowback efficiency is influenced by a variety of factors that vary over a wide range, and it is difficult to analyze the complex relationships between these parameters when discussing the effect of a particular factor on fracturing fluid flowback efficiency. The normalization approach is therefore an effective way of analyzing the results of the interaction of these factors. The capillary number, the dimensionless number proposed by Wilkinson ,31, is w−6, which are at very low values. In fact, at very low values of capillary number, capillary forces are dominant and the effect of other forces on fracturing fluid retention is modest [The capillary number for each experimental group is given in s modest , as can In this study, microscopic model experiments are performed to investigate the effects of fracturing fluid concentration and driving pressure difference on fracturing fluid flowback efficiency. The reticulated structure of fracturing fluid polymer molecules is analyzed from a microscopic perspective. The effects of viscous and capillary forces on fracturing fluid retention are discussed and the relationship between capillary number and fracturing fluid flowback efficiency is analyzed. Based on the experimental results, the following conclusions can be drawn.(1)In the early stages of fracturing fluid flowback, flowback efficiency increases rapidly and then plateaus. The ultimate fracturing fluid flowback efficiencies decreased as the concentration increased or the gas driving pressure differences decreased.(2)There are four kinds of mechanisms responsible for fracturing fluid retention in the pore network: viscous resistance, the Jamin effect, the gas blockage effect and the dead end of the pore. The viscous force and the capillary force are two important resistances to the fracturing fluid flowback process.(3)The ultimate flowback efficiency of the fracturing fluid increases linearly with increasing capillary number. At very low capillary numbers, capillary forces dominate fracturing fluid retention.


Accumulating evidence reveal that shorter courses of oral antibiotic therapy (OAT) may be an acceptable alternative, but immunocompromised hosts are frequently excluded from these studies. We evaluated outcomes in IV to OAT transitions for treating β-hemolytic streptococcal uncomplicated BSI (uBSI) in immunocompromised hosts.This retrospective cohort study evaluated immunocompromised adult patients hospitalized between 1/1/2013 and 12/31/2019 with uBSI due to β-hemolytic Streptococcus. Patients were excluded if source of infection was endovascular, central nervous system or bone/joint infection without definitive surgical intervention. We compared outcomes between IV only vs OAT cohorts. Primary outcome was 30-day all-cause mortality. Other outcomes included length of stay (LOS), BSI relapse, 30-day rehospitalization, and adverse drug events. The primary outcome was compared to previously presented findings in immunocompetent patients. Comparisons between groups were performed using Fisher’s exact test or Mann-Whitney test.Of 321 BSI screened, 52 immunocompromised adults with β-hemolytic Streptococcus uBSI identified. OAT was used in 25 episodes (48%). Cohort demographics were similar. PITT bacteremia score ≥4 was higher in the IV only cohort (30% vs. 0% p< 0.05). Immunocompromised categories primarily included patients with solid organ transplant/hematopoietic marrow transplant (44%) and heme/solid malignancy on chemotherapy (48%). Thirty-day mortality was lower in the OAT cohort (8% vs. 33% p< 0.05). There was no difference in LOS, readmission, BSI relapse or adverse events between cohorts (Table 1). There was no difference in 30-day mortality in OAT between immunocompromised and immunocompetent individuals (8% vs 2% p=0.15) despite overall increased 30-day mortality in immunocompromised patients (Table 2).Comparison of demographics and outcomes between OAT and IV only cohortsComparison of outcomes between immunocompromised and immunocompetent individualsOpportunities exist to modify management of uBSI in immunocompromised hosts. Transition to OAT did not increase 30-day mortality and was equivalent to immunocompetent hosts transitioned to OAT.Bryan T. Alexander, PharmD, BCIDP, AAHIVP, Astellas Pharma: Advisor/Consultant|F2G: Advisor/Consultant|Merck: Grant/Research Support Erica J. Stohs, MD, MPH, bioMerieux: Grant/Research Support|Merck: Grant/Research Support Trevor C. Van Schooneveld, MD, FSHEA, FACP, AN2 Therapeutics: Grant/Research Support|Biomeriuex: Advisor/Consultant|Biomeriuex: Grant/Research Support|Insmed: Grant/Research Support|Thermo-Fischer: Honoraria


A variant-adapted bivalent BNT162b2 mRNA vaccine  comprising original SARS-CoV-2 and Omicron BA.4/BA.5 spike proteins is authorized by the US FDA from 6 months of age as a primary series or as booster doses. We studied whether bivalent BNT162b2 booster generates improved immune responses against Omicron BA.4/BA.5 and ancestral strains and had a comparable safety profile to original BNT162b2 in 5–11-year-olds.This substudy is part of a phase 1/2/3 master study (NCT05543616) examining safety and immunogenicity of bivalent BNT162b2 in healthy children. The substudy group reported here is open label and evaluates a fourth dose with bivalent BNT162b2 10 μg  in 5–11-year-olds who previously received 3 original BNT162b2 10 μg doses. Reactogenicity (7 day), and 1 month safety and immunogenicity were assessed. SARS-CoV-2 Omicron BA.4/BA.5 and ancestral strain neutralization titers post dose 4 were descriptive immunogenicity endpoints. The comparator group for immunogenicity assessments included 113 participants from the initial pediatric study (NCT04816643) who received 3 original BNT162b2 10 μg doses and who were matched by age and SARS-CoV-2 infection status.Figure). No serious adverse events were reported. The safety and tolerability profile was generally consistent with that of original BNT162b2. Bivalent BNT162b2 elicited higher neutralizing titers against Omicron BA.4/BA.5 and similar titers against the ancestral strain 1 month post dose 4 compared with original BNT162b2 at 1 month post dose 3 overall and in those who were SARS-CoV-2 positive (Table).Of the 113 children who received bivalent BNT162b2, 50% were female, 58% White, and 58% SARS-CoV-2 positive at baseline. Median (range) time from dose 3 of original BNT162b2 to bivalent BNT162b2 was 5.5 (2.6−8.6) months. Bivalent BNT162b2 was well tolerated with mostly mild to moderate reactogenicity; no grade 4 events were observed  of bivalent BNT162b2 10 µg had a similar safety profile to original BNT162b2 10 µg and induced robust Omicron BA.4/BA.5 and ancestral strain neutralizing titers. These data support booster dosing with variant-adapted bivalent BNT162b2 in 5–11-year-olds.Grant C. Paulsen, MD, Moderna: Grant/Research Support|Pfizer: Grant/Research Support Lawrence Sher, MD, Pfizer Inc: Clinical Investigator Charu Sabharwal, MD, MPH, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Nicholas Kitchin, MD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Sungeen Hill, MD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Emily Wasserman, MD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Xia Xu, PhD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Yvonne A. Maldonado, MD, Pfizer: Grant/Research Support|Pfizer: Site Investigator, DSMB member Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Emmanuel Walter, MD, Clinetic: Clinical Investigator|Iliad Biotechnologies: Advisor/Consultant|Moderna: Clinical Investigator|Najit Technologies: Clinical Investigator|Pfizer Inc: Clinical Investigator|Sequiris: Clinical Investigator|Vaxcyte: Advisor/Consultant Flor M. Munoz, MD, MSc, CDC respiratory virus surveillance: Grant/Research Support|Gilead: Grant/Research Support|Moderna, sanofi, aztra zeneca, Merck, GSK: Advisor/Consultant|NIH: DSMB|NIH COVID-19 vaccines in pregnancy: Grant/Research Support|Pfizer Pediatric COVID-19 vaccines: Grant/Research Support|Pfizer, Dynavax, Monderna, Meissa, NIH: DSMB Eric Simoes, MD DCH, Abbott Diagnostics: Advisor/Consultant|Abbvie Inc: Advisor/Consultant|Abbvie Inc: DSMB study section|AstraZeneca: Grant/Research Support|AstraZeneca: travel|Bill and Melinda Gates Foundation: Advisor/Consultant|Bill and Melinda Gates Foundation: Grant/Research Support|Bill and Melinda Gates Foundation: travel, DSMB study section|CDC: Advisor/Consultant|CDC: travel|GSK plc: Advisor/Consultant|GSK plc: DSMB study section|Johnson & Johnson: Advisor/Consultant|Johnson & Johnson: Grant/Research Support|Merck & Co Inc: Grant/Research Support|National Institutes of Health: Grant/Research Support|National Institutes of Health: travel, DSMB study section|Novavax: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: travel|Regeneron: Grant/Research Support|Roche: Grant/Research Support|Roche: travel|USAID: Advisor/Consultant|USAID: Grant/Research Support|USAID: travel|WHO: Advisor/Consultant|WHO: travel Kawsar R. Talaat, MD, Intralytix: Advisor/Consultant|Merck: Advisor/Consultant|NIAID: DSMB|Pfizer: Grant/Research Support|Pfizer: Pfizer contract with institution|Sanofi: Grant/Research Support|Takeda: Advisor/Consultant Satoshi Kamidani, MD, CDC: Grant/Research Support|Emergent BioSolutions: Grant/Research Support|NIH: Grant/Research Support|Pfizer Inc: Grant/Research Support Lisa Moyer, BS, Pfizer: Employee|Pfizer: Stocks/Bonds Vrunda Parikh, PharmD, Pfizer: Employee|Pfizer: Stocks/Bonds Hua Ma, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Xingbin Wang, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Kenneth Koury, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Annaliesa S. Anderson, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Kena A. Swanson, Ph.D., Pfizer: Employee|Pfizer: Stocks/Bonds Alejandra C. Gurtman, M.D., Pfizer: Employee|Pfizer: Stocks/Bonds William C. Gruber, MD, Pfizer, Inc.: Employee|Pfizer, Inc.: Stocks/Bonds


IFNγ has long been recognised as a key mediator of tumour immunity and angiostasis. However, IFNγ modulation for cancer therapy is still unsuccessful due to its complex effects on various host cells. In this study, we found that treatment of Lewis lung carcinoma transplants with cisplatin often caused IFNγ-dependent tumour vascular damage. IFNγ induced endothelial glycolysis and lactate production, leading to enhanced endocytosis of vascular endothelial (VE)-cadherin and vessel leakage. We have also developed anti-IFNγ nanoparticles coated with a clot-binding peptide CREKA (CREKA-lipo-anti-IFNγ), which targets the fibrin-fibronectin complex that appears in the leaky site of damaged tumour blood vessels. Blocking IFNγ activity in the leakage site of capillaries using nanoparticles rescued VE-cadherin distribution on the endothelial cellular surface, promoted blood vessel integrity, and improved drug delivery. In conclusion, IFNγ blockade in capillary leak site protected tumour blood vessels from lactate-dependent VE-cadherin loss and enhanced drug delivery during chemotherapy, which provides a basis for tissue-specific IFNγ blockade for tumour therapy. Efficient drug penetration into solid tumours is a considerable challenge in cancer chemotherapy, which is one of the most important methods of clinical cancer treatment. For example, cisplatin [cis-diamminedichloridoplatinum (II)], a platinum-based anticancer chemotherapeutic drug, has long been used as a first-line drug in the treatment of lung cancer. Unfortunately, in most cases, chemotherapy only delays the development of the disease and has a 40-50% remission rate The integrity of blood vessels is regulated by endothelial metabolism IFNγ is an important modulator of tumour vascular functions In this study, we found that cisplatin-induced tumour vascular damage was largely dependent on IFNγ. IFNγ stimulation enhanced endothelial glycolysis and increased lactate production, which promoted endocytosis and degradation of VE-cadherin, leading to vascular leakage. We developed anti-IFNγ nanoparticles coated with CREKA peptide for the specific delivery of anti-IFNγ antibody to capillary leaky sites. The resulting nanoparticles, CREKA-lipo-anti-IFNγ, successfully rescued VE-cadherin distribution on endothelial cells and promoted chemotherapy. Our results provide a basis for improving tumour chemotherapy through tissue-specific IFNγ blockade.The mouse Lewis lung carcinoma (LLC) cell line and the mouse endothelial cell line sEND.1 cell line were cultured in a medium consisting of DMEM  supplemented with 10% foetal bovine serum .6) in 100 μL were subcutaneously (s.c.) injected into 6 to 8-week-old C57BL/6 female mice. Tumour growth was monitored every other day, and mortality was recorded daily during the entire experimental period. The lung cancer spontaneous mouse model  was a gift from Professor Xi Lin, Tsing Hua University. Mice were treated as previously described Female C57BL/6 wild-type (WT) mice were purchased from Vital River Laboratories . LLC cells  was injected i.p 6-8 h prior to euthanasia.Cisplatin  and anti-IFNγ prepared from hybridoma ATCC HB-170 as previously described Tumour tissues were harvested and embedded in optimal cutting temperature compound (OCT compound). They were then frozen and cut using a cryostat. For histological staining of tumour tissues, frozen sections (7 μm) were stained with Rat-anti-CD31 , Rabbit-anti-CD31 , Goat-anti-VE-cadherin , Rabbit-anti-NG2 , Rat-anti-CD146 , Rabbit-anti-GLUT1 , Rat-anti-cisplatin-modified DNA , and Rabbit-anti-γ-H2AX . Pictures were acquired using a Vectra, Perkin Elmer.+ blood vessels in tumour, the area of CD31+ blood vessels in each high-power field of tumour was analysed using Vectra, and the density of CD31+ blood vessels were presented as CD31+ area / total sectional area × 100%. Images used for analysis were derived from 2 to 3 tumours.For quantification of CD31+ blood vessels in tumour, colocalization of VE-cadherin and CD31 in each high-power field of tumour was analysed using Vectra, and the density of VE-cadherin+CD31+ blood vessels were presented as the area of colocalization / CD31+ area × 100%. Images used for analysis were derived from 2 to 3 tumours.For quantification of VE-cadherin+ area × 100%. Images used for analysis were derived from 3 tumours.For quantification of pericyte coverage in tumour, NG2  was used as the pericyte marker. NG2 is a marker of glial progenitor cells in the central nervous system, and has become the most common marker for pericytes. Pericyte specific deletion of NG2 leads to decreased pericyte ensheathment of endothelial cells, reduced vessel patency, and increased vessel leakiness + area in tumour, the area of GLUT1+ cells in each high-power field of tumour was analysed using Vectra, and GLUT1+ area was presented as GLUT1+ area/ total sectional area × 100%. Images used for analysis were derived from 3 tumours.For quantification of GLUT1+ cells in tumour after treatment, the number of Cpt+ cells in each high-power field of tumour was counted using Vectra, and the number of Cpt+ cells was presented as the number of Cpt+ cells / the number of DAPI+ cells × 100%. The quantification of γ-H2AX+ cells was similar to the quantification of Cpt+ cells. Images used for analysis were derived from 2 to 3 tumours.For quantification of Cpt+CD31+ area/ CD31+area × 100%.Fluorescein isothiocyanate (FITC)-lectin  was injected intravenously into tumour-bearing mice. Ten minutes later, mice were perfused by intracardiac injection of 1% paraformaldehyde (PFA) to remove circulating lectin, and tumour tissues were collected and embedded in OCT. Tumour sections (7 μm) were stained with Rat-anti-CD31  followed by an Alexa-Fluor-555 Goat-anti-Rat second antibody . Perfused blood vessels were calculated as FITC-lectinFITC-dextran  was administered intravenously to tumour-bearing mice. Mice were perfused by intracardiac injection of 1% PFA to remove circulating dextran ten minutes after injection, and tumours were harvested. The percentage of leaky vessels was measured as FITC-dextran area/total sectional area × 100%.IL-1β forward: TTCAGGCAGGCAGTATCACTC, reverse: GAAGGTCCACGGGAAAGACAC; IFNγ forward: ATGAACGCTACACACTGCATC, reverse: CCATCCTTTTGCCAGTTCCTC; TNFα forward: GACGTGGAACTGGCAGAAGAG, reverse: TTGGTGGTTTGTGAGTGTGAG; IL-6 forward: TAGTCCTTCCTACCCCAATTTCC, reverse: TTGGTCCTTAGCCACTCCTTC; IL-10 forward: GGTTGCCAAGCCTTATCGGA, reverse: ACCTGCTCCACTGCCTTGCT; IL-17 forward: TTTAACTCCCTTGGCGCAAAA, reverse: CTTTCCCTCCGCATTGACAC.To detect the mRNA expression of inflammatory factors in tumour tissues, total RNA was extracted from tumour tissues using TRIzol reagent  according to the manufacturer's instructions. First-strand cDNA was synthesised from 1 µg of total RNA using the Prime Script™ RT reagent kit . qPCR was performed using SYBR Premix Ex Taq II  and assessed using an Agilent Mx3005P instrument. The abundance of mRNA for each gene of interest was normalised to that of GAPDH. The primers used for the qPCR as follows: Mouse endothelial cells were isolated from tumours by using Tumor Dissociation Kit  and Tumor Cell Isolation Kit  according to the instructions, and then sorted by Flow cytometry by staning CD31. Isolated tumour endothelial cells were used for RNA sequencing. The data was analyzed by Z-Score normalization.DSPE-PEG-MAL  (8.5 mg) and CREKA (647 Da) peptide (1.78 mg) were dissolved in 4 mL of 10% methanol purged with nitrogen and stirred for 4 h at room temperature. The resulting product was dialysed against water for 24 h and then freeze-dried to obtain DSPE-PEG-CREKA and used as CREKA-lipo Liposomes loaded with anti-IFNγ antibody (CREKA-lipo-anti-IFNγ) were prepared using the double emulsion-solvent evaporation method -anti-IFNγ and CREKA-Lipo were assessed by dynamic light scattering (DLS) using the ZetaSizer Nano series Nano-ZS .The size distribution and zeta potential of CREKA-Lipo-anti-IFNγ was maintained in dialysis bag (molecular weight cutoff of 300 kD), and placed in 15 mL PBS  buffer in a 50 mL tube, general shaking at 37ºC. At certain time points, 1 mL external buffer was taken and amount of anti-IFNγ was detected by UV-spectrophotometer at 280 nm. At the same time, 1 mL fresh PBS was added into the tube to maintain a consistent total volume. The accumulative release of anti-IFNγ was calculated.In order to detect the drug release profiles, CREKA-LipoMice (6-8 weeks old) were intravenously injected with CREKA-lipo-anti-IFNγ (2.5 mg/kg) or CREKA-lipo. Eight days after treatment, blood serum was collected from the orbital sinus, and concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT) (associated with liver function), and creatinine (CR) (associated with kidney function) were measured using the AST/ALT/CR Detection Kit (Servicebio) following the manufacturer's guidance. The animals were sacrificed, and the mouse heart, liver, spleen, lung, and kidney were collected from each mouse for Hematoxylin and Eosin (H&E) staining to evaluate their morphological changes.sEND.1 cells  dispensed in 80 µL culture medium per well were seeded into XF96 Cell Culture Microplates (Agilent). After overnight culture, cells were treated with or without IFNγ  and AZD5363  for 6 h. The culture medium was changed to XF Base Medium (Agilent) containing 1 mM L-glutamine 1 h before starting the assay. Glucose, oligomycin, and 2-deoxy-D-glucose (2-DG) were subsequently injected into the medium at final concentrations of 10 mM, 1 μM, and 50 mM, respectively. The extracellular acidification rate (ECAR) was measured using a Seahorse XF96 extracellular-flux analyser (Agilent). Finally, we used Hoechst 33342  to normalise the cell numbers while following the manufacturer's instructions.sEND.1 cells were harvested and lysed with RIPA buffer  supplemented with 1 μM phenylmethylsulfonyl fluoride  and protease inhibitor cocktail . Cells were then collected and lysed with RIPA buffer for the western blot assay. The Membrane and Cytosol Protein Extraction Kit  was used to extract the cell membrane and cytosol proteins. Quantified protein lysates were measured with the Protein BCA Assay Kit  according to the manufacturer's instructions. Proteins in lysates were resolved on SDS-PAGE gels, transferred onto polyvinylidene fluoride membranes , and immunoblotted with Rabbit-anti-GLUT1 , Rabbit-anti-HK2 , Rabbit-anti-p-PFKFB2 , Rabbit-anti-PFKFB2 , Rabbit-anti-p-AKT , Mouse-anti-AKT , Rabbit-anti-p-ERK , Rabbit-anti-ERK , Rabbit-anti-p-P38 , Mouse-anti-P38 , Goat-anti-VE-cadherin , and Mouse-anti-β-actin . Protein bands were visualised using an eECL Western Blot Kit . Signal intensities were determined using Quantity One Image Analyser .sEND.1 cells were cultured in confocal dishes  overnight and treated with or without sodium lactate , IFNγ , Lactate dehydrogenase A (LDHA) inhibitor , and bafilomycin  for 12 h. Cells were then fixed with 4% paraformaldehyde for 15 min and permeabilised with 0.5% Triton X-100 for 5 min. Next, cells were blocked with 3% (w/v) BSA in phosphate buffered saline (PBS) for 30 min at 37 °C and then incubated with Goat-anti-VE-cadherin  and Rabbit-anti-LAMP  for 1 h. Next, these were labelled with Alexa-Fluor-555 donkey-anti-goat second antibody  and Alexa-Fluor-488 donkey anti-rabbit second antibody  for 50 min at 37 °C, and stained with 4',6-diamidino-2-phenylindole  for 10 min. The images were captured using a spinning-disk confocal microscope.2-[N-amino]-2-deoxyglucose (2-NBDG) is taken up into cells through glucose transporters and phosphorylated by hexokinase. sEND.1 cells were seeded in a 24 well plate. After overnight culture, cells were treated with or without IFNγ  and AZD5363  for 12 h. The culture medium was discarded and cells were washed with PBS (pH 7.4). This was followed by the addition of low-glucose culture media supplemented with 2-NBDG  and an incubation of 45 min at 37 °C. Next, cells were washed with PBS and trypsin was added, and they were harvested, centrifuged at 1,500 rpm for 5 min at 4 °C, washed twice with ice-cold PBS, and kept on ice. A control sample lacking 2-NBDG was used to set the blank in the flow cytometer and gate parameters for 2-NBDG detection.sEND.1 cells were seeded in a 6 well plate. After overnight culture, cells were treated with or without IFNγ  and AZD5363  for 12 h. The cells were then collected to detect lactate production using a lactate colorimetric/fluorometric assay kit  while following the manufacturer's instructions.sEND.1 Cells were seeded onto the upper chamber of the transwell system . When the cells were confluent in the upper chambers, they were stimulated with or without sodium lactate , IFNγ  and LDHA inhibitor  for 48 h. Thirty minutes before harvest, FITC-dextran  were added to the upper chamber and incubated at 37 °C. Finally, 100 µL of liquid was aspirated into the lower chamber, and the fluorescence of FITC-dextran was measured using a microplate reader.p < 0.05, ** p < 0.01, and *** p < 0.001.Statistical analyses using Nonparametric Mann-Whitney test or two-way ANOVA were performed using GraphPad Prism software . Differences were considered significant when P values were less than 0.05. Statistically significant differences are indicated as follows: * 3Figure A. Structural and functional changes in tumour blood vessels were analysed by immunostaining for CD31, VE-cadherin, and NG2 four days after the cessation of chemotherapy . In the microcirculation, NG2 identifies perivascular cells along capillaries, and represents the mature and functional blood vessels + tumour blood vessels, the distribution of VE-cadherin in endothelial cells when compared with those in the non-treated tumour, while NG2+ pericyte coverage has no difference between the two groups Figure B. These results indicated that cisplatin induced structural damage to the tumour blood vessels and impaired vascular integrity. To measure the vascular perfusion and permeability after cisplatin treatment, FITC-labelled lectin and dextran were injected intravenously into tumour-bearing mice. Cisplatin treatment reduced vascular perfusion, but markedly promoted dextran leakage in the tumour Figure C.A mouse lung cancer model was used to examine the possibility of cisplatin treatment impairing vascular perfusion. LLC cells were subcutaneously transplanted into wild-type (WT) C57Bl/6 mice. Cisplatin (1 mg/kg) was injected every other day, four times, when the tumour volume reached ~200 mmFigure D. Consistently, the Cytometric Bead Array (CBA) results confirmed that cisplatin induced high expression of IFNγ two days after cisplatin treatment Figure E.Cisplatin might directly damage endothelial cells or indirectly influence endothelial cells through activation of the immune response Figure A. Based on the observation of ~10 pg/g IFNγ in the tumour tissues  + tumour blood vascular density Figure B and the distribution of VE-cadherin on tumour vessels Figure B. Although cisplatin treatment did not reduce the NG2+ pericyte coverage compared with control, anti-IFNγ increased NG2+ pericyte coverage compared with cisplatin-treated tumours Figure B. Importantly, anti-IFNγ combined with cisplatin treatment increased the perfusion of lectin into the tumour vessels Figure C while reducing the leakage of dextran into the tumour vessels Figure C. The proportion of reduced vascular leakage was about 60%  Figure C. Finally, the GLUT1+ area, that reflects hypoxia in tumour tissues  Figure D, implying that anti-IFNγ might alleviated hypoxia in the tumour. Taken together, these results indicate that cisplatin-induced vascular damage is largely dependent on the increased expression of IFNγ after chemotherapy, and that neutralising IFNγ could improve vascular function.Next, IFNγ was neutralised to explore whether the cisplatin-induced vascular damage was dependent on IFNγ. The IFNγ neutralising antibody was administered two days after the first cisplatin injection in the transplanted LLC tumour models Figure A, B.Endothelial cell metabolism is crucial for vascular function Supplementary Figure S1). Moreover, IFNγ promoted the uptake of glucose in sEND.1 cells by measuring 2-NBDG fluorescence intensity Figure C. Furthermore, IFNγ increased the expression of GLUT1, HK2 and PFKFB2, which are the key enzymes involved in glycolysis Figure D. Lactate is the final product of the glycolysis pathway and IFNγ promoted the production of lactate Figure E. We isolated endothelial cells from mouse tumour tissues for RNA sequencing. The results revealed that cisplatin increased the expressions of glycolysis-related genes compared with control, including PFKFB2 and HK2. Anti-IFNγ combined with cisplatin reduced the expressions of these genes compared with cisplatin alone . Moreover, elevated glucose uptake induced by IFNγ was reduced by the AKT inhibitor Figure I. Furthermore, inhibition of AKT signalling downregulated IFNγ-promoted protein levels of GLUT1, HK2 and p-PFKFB2 Figure J. Lactate is the product of glycolysis, and IFNγ-induced increased lactate levels could be reduced by inhibition of AKT Figure K. Taken together, these results demonstrated that IFNγ increased glycolysis of endothelial cells by activating the AKT signalling pathway.To further investigate how IFNγ increased glycolysis of endothelial cells, sEND.1 cells were cultured with or without IFNγ Figure A. Inhibition of LDHA that catalyses pyruvate to lactate by GNE140 during glycolysis, reversed IFNγ-promoted endocytosis of VE-cadherin in endothelial cells Figure B. Moreover, lactate reduced the expression of VE-cadherin on the membrane while increasing intracellular VE-cadherin levels Figure C. IFNγ had the same effect on VE-cadherin; however, inhibiting LDHA diminished the effect of IFNγ Figure D. A permeability assay was performed to detect whether lactate promotes leakage of endothelial cells. The results showed that lactate promoted the leakage of endothelial cells Figure E. Inhibition of LDHA reversed the IFNγ-induced increase in the leakage of endothelial cells Figure F. These findings demonstrated that IFNγ-induced lactate reduced the distribution of VE-cadherin on the membrane, promoted endocytosis of VE-cadherin, and enhanced the degradation of intracellular VE-cadherin by activating lysosomes.VE-cadherin plays an important role in maintaining vascular integrity. VE-cadherin on the membrane connects gaps between endothelial cells and maintains the integrity of the blood vessels, while internalised VE-cadherin could either be recycled in the cell membrane to maintain VE-cadherin integrity or degraded in lysosomes, which leads to endothelial cell barrier dysfunction Figure A. When anti-IFNγ antibody was administered two days before the first injection of cisplatin, it did not affect the outcome of chemotherapy Figure A. However, when anti-IFNγ antibody was administered simultaneously with cisplatin, it impaired the beneficial effects of chemotherapy Figure A, which was consistent with the observation that IFNγ might be involved in anti-tumour immunity during chemotherapy. When anti-IFNγ antibody was administered two days after the first injection of cisplatin, it largely promoted tumour chemotherapy Figure A, B.Destruction of tumour blood vessels reduces the osmotic concentration of chemotherapeutic drugs in the tumour tissue, thereby weakening the effect of killing tumour cell, leading to chemotherapy resistance Figure B and tumour weight Figure C to a greater degree compared than that reduced by cisplatin alone. Moreover, neutralising IFNγ combined with cisplatin significantly increased the overall survival of mice compared that increased by cisplatin alone Figure D. Cpt-DNA adducts within tumour tissues were increased by anti-IFNγ treatment and the effective rate of drug delivery was increased by about 113%  Figure E, as shown by immunostaining. Cisplatin causes DNA cross-linking and stimulates H2AX phosphorylation at serine 139 to generate γ-H2AX as a major marker for DNA damage signalling in response to DNA double-strand breaks + area in the tumour tissue was increased by neutralising IFNγ treatment Figure F. These results indicated that neutralising IFNγ enhanced the delivery of cisplatin into the tumour, thereby promoting the chemotherapeutic effect.We further explored effects of neutralising IFNγ after the first cisplatin injection. Neutralising IFNγ combined with cisplatin reduced tumour volume L858R; CCSP-rtTA). After sustained induction by doxycycline (2 mg/mL) for three months, cisplatin was injected every other day for four times. The IFNγ neutralising antibody was given two days after the first cisplatin injection. During this period, doxycycline was continually administered Figure G. First, pericyte coverage was detected by examining the expression of NG2+CD146+ tumour blood vessels by immunofluorescence analysis and found that anti-IFNγ increases NG2+ pericyte coverage compared with that observed in cisplatin-treated tumours , thus implying that compared with cisplatin, anti-IFNγ enhances the integrity of tumour blood vessels. Then tumour nodes in the lungs were counted. In this model, cisplatin treatment did not significantly reduce the number of tumour nodes in the lung compared with that in the control. Neutralisation of IFNγ combined with cisplatin significantly reduced the number of lung nodes compared with that by cisplatin treatment alone Figure H.In addition to the lung cancer xenograft mouse model, we constructed a lung cancer spontaneous mouse model . Then the characterization detection of CREKA-lipo-anti-IFNγ was performed. We have determined the diameter, potential and release profiles of the CREKA-lipo-anti-IFNγ nanoparticle. Dynamic light scattering analysis showed that the average hydrodynamic diameter of CREKA-Lipo-anti-IFNγ was about 316 nm, which is larger than the diameter of empty nanoparticles  (Supplementary ). In response to the addition of anti-IFNγ, the average zeta potential of the particles increased from -0.675 mV to -0.275 mV (Supplementary ), thereby indicating that drug loading affected the net charge of the nanoparticles. To evaluate the drug release profiles of CREKA-Lipo-anti-IFNγ, the accumulative drug release at both pH 6.5 and 7.4 was investigated. The result showed that CREKA-Lipo-anti-IFNγ is characterized by a relatively more rapid drug release at pH 6.5 than at pH 7.4. At pH 6.5, we detected approximately 43% accumulative drug release at 48 h (Supplementary ). Because acidic condition enhances the release of drugs by CREKA-lipo nanoparticles Supplementary ), in consistence with previous study Figure A. We found that CREKA-lipo-anti-IFNγ combined with cisplatin treatment increased the density of CD31+ tumour blood vessels and the distribution of endothelial junctional molecule VE-cadherin when compared with those by cisplatin alone, while lactate reversed the effects of CREKA-lipo-anti-IFNγ on blood vessels Figure B. Moreover, DNA damage indicated by γ-H2AX staining in tumour tissues was increased by CREKA-lipo-anti-IFNγ treatment, which was inhibited by lactate treatment Figure C. CREKA-lipo-anti-IFNγ combined with cisplatin reduced tumour volume compared with those reduced by cisplatin alone, while lactate abrogated the effect of CREKA-lipo-anti-IFNγ Figure D. CREKA-lipo-anti-IFNγ combined with cisplatin reduces tumour weight compared with that in control group mice, whereas lactate abrogates the effect of CREKA-lipo-anti-IFNγ Figure E. In summary, these results demonstrated that the delivery of IFNγ blockade to the capillary leak site prevented cisplatin-induced vascular damage, thereby promoting drug delivery and improving the chemotherapy effect Figure .The outcome of systematic application of anti-IFNγ antibodies is difficult to control because of various targets. We sought to locally deliver anti-IFNγ antibodies to the capillary leak site during chemotherapy. We coated nanoparticles with CREKA and loaded anti-IFNγ antibodies into nanoparticles (CREKA-lipo-anti-IFNγ). The tumour-homing pentapeptide (CREKA) on the surface of nanoparticles specifically targets fibrin-fibronectin complexes within the microthrombi that are found on the walls of leaky tumour blood vessels. We confirmed that the tumour blood vessels were enwrapped by fibronectin in the LLC tumour with cisplatin treatment (IFNγ is believed to be a central mediator of anti-tumour immunity + T lymphocytes or CD8+ T lymphocytes to produce IFNγ, thereby increasing tumour blood vessel normalisation Inhibition of IFNγ around leaky blood vessels is an effective way to promote vascular integrity and drug penetration in tumour IFNγ-induced internalisation of VE-cadherin is a major mechanism of cisplatin-induced vascular leakage in tumours. Inflammatory cytokines play an important role in chemotherapy-induced vascular damage in tumours. These results revealed that tumour blood vessels were damaged after cisplatin treatment. Tumour cells are more sensitive than endothelial cells to cisplatin treatment IFNγ stimulates the internalisation of VE-cadherin in endothelial cells by modulating endothelial glycolysis. Our findings showed that the IFNγ-stimulated loss of VE-cadherin on endothelial cells was dependent on lactate production. IFNγ increased endothelial glycolysis and lactate production by activating AKT signalling. AKT is a signalling pathway that primarily promotes glycolysis IFNγ blockade has the potential to prevent chemotherapy-associated lethal side effects. Chemotherapy treatment-associated side effects are a cause of death in patients with cancer. In cancer patients, chemotherapeutic drugs have been reported to cause capillary leak syndrome In summary, we demonstrate that IFNγ blockade in capillary leak site prevents drug-induced vascular damages, improves drug delivery and chemotherapeutic efficacy in mouse lung cancer models. Cisplatin treatment induces the increase of IFNγ, which promotes the endocytosis of VE-cadherin through instigating endothelial glycolysis and lactate production. Neutralizing IFNγ maintains VE-cadherin on endothelial membrane and guarantees vascular integrity, providing an efficient tunnel for drug delivery. Our results provide a basis for tissue-specific delivery of IFNγ blockade for the improvement of tumor chemotherapy.Supplementary figures.Click here for additional data file.


Fusarium solani species complex fungal DNA was identified in cerebral spinal fluid (CSF) by polymerase chain reaction and next-generation metagenomic sequencing; a highly resistant strain was isolated from a single tissue culture. We describe epidemiology, demographics, and clinical features to inform prevention messaging, guidance, and future responses.US public health officials are responding to a multinational, multistate fungal meningitis outbreak involving travelers who had primarily cosmetic procedures  under epidural anesthesia at 2 Matamoros, Mexico clinics (closed 5/13/2023 by Mexican authorities). Public health officials collected data on confirmed and probable cases (1/1/23–7/25/2023) using a standardized case report form; data were analyzed in Microsoft Excel.In total, 10 probable (1 death) and 10 confirmed (8 deaths) US fungal meningitis cases were identified; all involved epidural anesthesia from the same anesthesiologist at both clinics. Most cases (80%) occurred in Texas residents. Median patient age was 31 years (range: 23–52); 19 patients were women; 13 were Hispanic/Latino. None had underlying conditions reported. At least 4 patients were uninsured, with several reporting associated care-seeking delays. Common symptoms were headache (n=18), nausea (n=13), fever (n=12), and stiff neck (n=12). Median (IQR) initial CSF results were white blood cell count (498/µL [343–825]), glucose (31 mg/dL [26–40]), and protein (99 mg/dL [52–2140]). In 8 patients, CSF -β-D-Glucan was ≥ 500 pg/ml. From procedure date, average days (range) to symptom onset was 19 (0–58), to hospitalization was 52 (14–106).In the US, the fungal meningitis outbreak has primarily affected young, healthy Hispanic/Latino women travelling to Matamoros, Mexico, for procedures and resulted in a high mortality rate (40%). CDC advises US residents against having elective procedures in Matamoros involving epidural anesthetic injection. Insurance barriers may have led to medical tourism to Mexico and subsequent delayed treatment for fungal meningitis, highlighting the need for preparation to address healthcare access disparities in future outbreaks.Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA, FECMM, CMQ, Astellas: Advisor/Consultant|Astellas: Grant/Research Support|Astellas: Honoraria|F2G: Advisor/Consultant|F2G: Grant/Research Support|F2G: Honoraria|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Gilead: Honoraria|GSK: Advisor/Consultant|GSK: Grant/Research Support|GSK: Honoraria|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Melinta: Honoraria|NIH: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Expert Testimony|Pfizer: Honoraria|Pulmocide: Advisor/Consultant|Pulmocide: Grant/Research Support|Pulmocide: Honoraria|Scynexis: Advisor/Consultant|Scynexis: Grant/Research Support|Scynexis: Honoraria|T2 biosystems: Advisor/Consultant|T2 biosystems: Grant/Research Support|T2 biosystems: Honoraria|Viracor: Advisor/Consultant|Viracor: Grant/Research Support|Viracor: Honoraria


Pseudomonas aeruginosa is complex and multifaceted. While the novel β-lactamase inhibitors (BLI), avibactam, relebactam, and vaborbactam inhibit serine-based β-lactamases, the comparative potency of the novel β-lactam (BL) /BLI combinations against serine-carbapenemase producing P. aeruginosa is unknown. The present study sought to compare the in vitro activity of ceftazidime/avibactam, ceftazidime, imipenem/relebactam, imipenem, meropenem/vaborbactam, and meropenem against serine-β-lactamase producing P. aeruginosa.Antimicrobial resistance in P. aeruginosa were collated through the Enhancing Rational Antimicrobials against Carbapenem-resistant P. aeruginosa (ERACE-PA) Global Surveillance. Isolates positive for serine-based carbapenemases were assessed. Minimum inhibitory concentrations (MICs) were determined by broth microdilution to each novel-BL/BLI and BL alone. In vitro potency was assessed by MIC50/90 and percent of isolates susceptible per CLSI guidance.Carbapenem-resistant 50/MIC90 values of 4/8 mg/L and 91% of isolates were susceptible. Conversely, ceftazidime alone was active against only 3% of isolates. The MIC50/MIC90 of imipenem/relebactam were 16/ >16 mg/L and 13% of all isolates were defined as susceptibility. Against the KPC-producing isolates, 38% were susceptible to imipenem/relebactam compared with 0% for imipenem. The meropenem/vaborbactam MIC50/MIC90 were >16/ >16 mg/L, and 6% of isolates were susceptible which was similar to meropenem alone  suggesting the addition of vaborbactam cannot overcome co-expressed, non-enzymatic resistance mechanisms.GES was the most common carbapenemase identified (n=59) followed by KPC (n=8). Ceftazidime/avibactam had MICin vitro activity and thus is a rational treatment option for serine-carbapenemase harboring P. aeruginosa. While imipenem/relebactam displayed some activity particularly against isolates with KPCbla, meropenem/vaborbactam exhibited poor activity with MICs similar to meropenem alone. These data can be integrated with rapid molecular diagnostics to guide empiric therapy.Among the novel BL/BLIs, ceftazidime/avibactam displayed better Christian M. Gill, PharmD, Cepheid: Grant/Research Support|Entasis therapeutics: Grant/Research Support|Everest Medicines: Grant/Research Support|Shionogi: Grant/Research Support David P. Nicolau, PharmD, Allergan: Advisor/Consultant|Allergan: Grant/Research Support|Cepheid: Advisor/Consultant|Cepheid: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Tetraphase: Advisor/Consultant|Tetraphase: Grant/Research Support|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant|Wockhardt: Grant/Research Support