Individual variation in the emergence of anterior-to-posterior neural fates from human pluripotent stem cells.

Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development.

Antisera against flagellin A or B inhibits Pseudomonas aeruginosa motility as measured by novel video microscopy assay.

Flagellum-mediated motility is essential to Pseudomonas aeruginosa (P. aeruginosa) virulence. Antibody against flagellin reduces motility and inhibits the spread of the bacteria from the infection site. The standard soft-agar assay to demonstrate anti-flagella motility inhibition requires long incubation times, is difficult to interpret, and requires large amounts of antibody. We have developed a time-lapse video microscopy method to analyze anti-flagellin P. aeruginosa motility inhibition that has several advantages over the soft agar assay. Antisera from mice immunized with flagellin type A or B were incubated with Green Fluorescent Protein (GFP)-expressing P. aeruginosa strain PAO1 (FlaB+) and GFP-expressing P. aeruginosa strain PAK (FlaA+). We analyzed the motion of the bacteria in video taken in ten second time intervals. An easily measurable decrease in bacterial locomotion was observed microscopically within minutes after the addition of small volumes of flagellin antiserum. From data analysis, we were able to quantify the efficacy of anti-flagellin antibodies in the test serum that decreased P. aeruginosa motility. This new video microscopy method to assess functional activity of anti-flagellin antibodies required less serum, less time, and had more robust and reproducible endpoints than the standard soft agar motility inhibition assay.

Photobiomodulation therapy using a class 3b laser does not cause premature rupture of multivesicular liposomal bupivacaine suspensions in a cadaver model.

OBJECTIVE: To determine if photobiomodulation causes a premature release of liposomal bupivacaine (LB) suspensions. ANIMALS: A 25-kg mixed breed dog cadaver euthanized for reasons unrelated to this study. METHODS: In September 2022, a proximomedial tibial incision was made in a dog cadaver, and a tibial plateau leveling osteotomy plate was implanted. A stab incision was made one-half inch distal to the incision, and a tunnel to the plate was created prior to closure of the primary wound. A 3-cc anal sac catheter was advanced through the distal incision until the bulb rested against the face of the plate. Seven treatment groups of treatment power (watts) and total energy (joules/cm2) were defined as: A, 0.5 W, 2.0 J/cm2; B, 0.5 W, 4.0 J/cm2; C, 0.5 W, 6.0 J/cm2; D, 1.0 W, 2.0 J/cm2; E, 1.0 W, 4.0 J/cm2; F, 1.0 W, 6.0 J/cm2; and sham, 0.0 W, 0.0 J/cm2. Ten samples per group of 2 mL of LB were infused into a new catheter and treated percutaneously with a class 3b laser. All samples remained in the catheter for 12 seconds to reflect the longest treatment time. Post-treatment free bupivacaine concentrations were identified with high-performance liquid chromatography. RESULTS: The median free bupivacaine concentration was reported as: sham, 1.89 mg/mL; A, 1.93 mg/mL; B, 2.01 mg/mL; C, 2.05 mg/mL; D, 1.92 mg/mL; E, 2.03 mg/mL; and F, 2.00 mg/mL. There were no differences in median free bupivacaine concentrations between groups (P = .988). CLINICAL RELEVANCE: Concurrent LB and photobiomodulation are recommended during the postoperative period. The results of this proof-of-concept study suggest that concurrent use of LB and photobiomodulation may be safe, but in vivo studies at similar and stronger photobiomodulation settings are warranted.

Antibody responses in Klebsiella pneumoniae bloodstream infection: a cohort study.

Background: Klebsiella pneumonia (Kpn) is the fourth leading cause of infection-related deaths globally, yet little is known about human antibody responses to invasive Kpn. In this study, we sought to determine whether the O-specific polysaccharide (OPS) antigen, a vaccine candidate, is immunogenic in humans with Kpn bloodstream infection (BSI). We also sought to define the cross-reactivity of human antibody responses among structurally related Kpn OPS subtypes and to assess the impact of capsule production on OPS-targeted antibody binding and function. Methods: We measured plasma antibody responses to OPS (and MrkA, a fimbrial protein) in a cohort of patients with Kpn BSI and compared these with controls, including a cohort of healthy individuals and a cohort of individuals with Enterococcus BSI. We performed flow cytometry to measure the impact of Kpn capsule production on whole cell antibody binding and complement deposition, utilizing patient isolates with variable levels of capsule production and isogenic capsule-deficient strains derived from these isolates. Findings: We enrolled 69 patients with Kpn BSI. Common OPS serotypes accounted for 57/69 (83%) of infections. OPS was highly immunogenic in patients with Kpn BSI, and peak OPS-IgG antibody responses in patients were 10 to 30-fold higher than antibody levels detected in healthy controls, depending on the serotype. There was significant cross-reactivity among structurally similar OPS subtypes, including the O1v1/O1v2, O2v1/O2v2 and O3/O3b subtypes. Physiological amounts of capsule produced by both hyperencapsulated and non-hyperencapsulated Kpn significantly inhibited OPS-targeted antibody binding and function. Interpretation: OPS was highly immunogenic in patients with Kpn BSI, supporting its potential as a candidate vaccine antigen. The strong cross-reactivity observed between similar OPS subtypes in humans with Kpn BSI suggests that it may not be necessary to include all subtypes in an OPS-based vaccine. However, these observations are tempered by the fact that capsule production, even in non-highly encapsulated strains, has the potential to interfere with OPS antibody binding. This may limit the effectiveness of vaccines that exclusively target OPS. Funding: National Institute of Allergy and Infectious Diseases at the National Institutes of Health. Research in Context: Evidence before this study: Despite the potential of O-specific polysaccharide (OPS) as a vaccine antigen against Klebsiella pneumoniae (Kpn), the immunogenicity of OPS in humans remains largely unstudied, creating a significant knowledge gap with regard to vaccine development. A search of PubMed for publications up to March 18, 2024, using the terms " Klebsiella pneumoniae " and "O-specific polysaccharide" or "O-antigen" or "lipopolysaccharide" revealed no prior studies addressing OPS antibody responses in humans with Kpn bloodstream infections (BSI). One prior study 1 evaluated antibody response to a single lipopolysaccharide (which contains one subtype of OPS) in humans with invasive Kpn infection; however, in this study OPS typing of the infecting strains and target antigen were not described. Added value of this study: Our investigation into OPS immunogenicity in a human cohort marks a significant advance. Analyzing plasma antibody responses in 69 patients with Kpn BSI, we found OPS to be broadly immunogenic across all the types and subtypes examined, and there was significant cross-reactivity among structurally related OPS antigens. We also demonstrated that Kpn capsule production inhibit OPS antibody binding and the activation of complement on the bacterial surface, even in classical Kpn strains expressing lower levels of capsule.Implications of all the available evidence: While the immunogenicity and broad cross-reactivity of OPS in humans with Kpn BSI suggests it is a promising vaccine candidate, the obstruction of OPS antibody binding and engagement by physiologic levels of Kpn capsule underscores the potential limitations of an exclusively OPS-antigen based vaccine for Kpn. Our study provides insights for the strategic development of vaccines aimed at combating Kpn infections, an important antimicrobial resistant pathogen.

Vaccine value profile for Klebsiella pneumoniae.

Klebsiella pneumoniae causes community- and healthcare-associated infections in children and adults. Globally in 2019, an estimated 1.27 million (95% Uncertainty Interval [UI]: 0.91-1.71) and 4.95 million (95% UI: 3.62-6.57) deaths were attributed to and associated with bacterial antimicrobial resistance (AMR), respectively. K. pneumoniae was the second leading pathogen in deaths attributed to AMR resistant bacteria. Furthermore, the rise of antimicrobial resistance in both community- and hospital-acquired infections is a concern for neonates and infants who are at high risk for invasive bacterial disease. There is a limited antibiotic pipeline for new antibiotics to treat multidrug resistant infections, and vaccines targeted against K. pneumoniae are considered to be of priority by the World Health Organization. Vaccination of pregnant women against K. pneumoniae could reduce the risk of invasive K.pneumoniae disease in their young offspring. In addition, vulnerable children, adolescents and adult populations at risk of K. pneumoniae disease with underlying diseases such as immunosuppression from underlying hematologic malignancy, chemotherapy, patients undergoing abdominal and/or urinary surgical procedures, or prolonged intensive care management are also potential target groups for a K. pneumoniae vaccine. A 'Vaccine Value Profile' (VVP) for K.pneumoniae, which contemplates vaccination of pregnant women to protect their babies from birth through to at least three months of age and other high-risk populations, provides a high-level, holistic assessment of the available information to inform the potential public health, economic and societal value of a pipeline of K. pneumoniae vaccines and other preventatives and therapeutics. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public-private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the WHO. All contributors have extensive expertise on various elements of the K.pneumoniae VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.