IgG hexamers initiate complement-dependent acute lung injury.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, in reactions to transfusions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving the assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was not clear, so we tested their relevance in a mouse model of alloantibody- and complement-mediated acute lung injury. We used 3 approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer "decoy" therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

IgG hexamers initiate acute lung injury.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. A previously overlooked step in complement activation by IgG antibodies has been elucidated involving interactions between IgG Fc domains that enable assembly of IgG hexamers, which can optimally activate the complement cascade. Here, we tested the in vivo relevance of IgG hexamers in a complement-dependent alloantibody model of acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer 'decoy' therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate a direct in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

Optimizing anesthesia and delivery approaches for dosing into lungs of mice.

Microbes, toxins, therapeutics, and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing in mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52 ± 9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30 ± 15%). This difference in pulmonary dose delivery altered key outcomes in models of viral and bacterial pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus or Pseudomonas aeruginosa developing more robust lung inflammation responses relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63 ± 8% of dose to lungs, and a nonsurgical intratracheal dosing approach further increased lung delivery to 92 ± 6% of dose. The use of either of these more precise dosing methods yielded greater experimental power in the bacterial pneumonia model relative to intranasal infection. Both anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors affect experimental power and so should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.NEW & NOTEWORTHY Many lung research studies involve dosing fluids into lungs of mice. In this study, the authors measure lung deposition using intranasal (i.n.), oropharyngeal aspiration (o.a.), and intratracheal (i.t.) dosing methods in mice. Anesthetic approach and administration route were found to affect pulmonary dosing efficiency. The authors demonstrate that refinements to dosing techniques can enable reductions in the number of animals needed for bacterial and viral pneumonia studies.

Biophysical Changes of Leukocyte Activation (and NETosis) in the Cellular Host Response to Sepsis.

Sepsis, the leading cause of mortality in hospitals, currently lacks effective early diagnostics. A new cellular host response test, the IntelliSep test, may provide an indicator of the immune dysregulation characterizing sepsis. The objective of this study was to examine the correlation between the measurements performed using this test and biological markers and processes associated with sepsis. Phorbol myristate acetate (PMA), an agonist of neutrophils known to induce neutrophil extracellular trap (NET) formation, was added to whole blood of healthy volunteers at concentrations of 0, 200, and 400 nM and then evaluated using the IntelliSep test. Separately, plasma from a cohort of subjects was segregated into Control and Diseased populations and tested for levels of NET components (citrullinated histone (cit-H3) DNA and neutrophil elastase (NE) DNA) using customized ELISA assays and correlated with ISI scores from the same patient samples. Significant increases in IntelliSep Index (ISI) scores were observed with increasing concentrations of PMA in healthy blood (0 and 200: p < 10-10; 0 and 400: p < 10-10). Linear correlation was observed between the ISI and quantities of NE DNA and Cit-H3 DNA in patient samples. Together these experiments demonstrate that the IntelliSep test is associated with the biological processes of leukocyte activation and NETosis and may indicate changes consistent with sepsis.

Optimizing anesthesia and delivery approaches for dosing into lungs of mice.

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in models of viral and bacterial pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus or Pseudomonas aeruginosa developing more robust lung inflammation responses relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. Use of either of these more precise dosing methods yielded greater experimental power in the bacterial pneumonia model relative to intranasal infection. Both anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors affect experimental power and so should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.

Sepsis promotes splenic production of a protective platelet pool with high CD40 ligand expression.

Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen was a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen, where IL-3 induced their differentiation into megakaryocytes (MKs). In the spleen, immune-skewed MKs produced a CD40 ligandhi platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.

Elements of Regulatory Dissonance: Examining FDA and EMA Product Labeling of New Vaccines (2006-2018).

With the ongoing globalization of the pharmaceutical industry, efforts to harmonize technical requirements of registering drugs and biologics, including vaccines, have produced a number of useful guidelines utilized around the world. However, such efforts have not been extended to the regulatory review process or product labeling. Prescribing information and patient information leaflet are two types of such product labeling documents. This study examined the differences in the languages of these documents between the United States (US) and European Union (EU). The key documents examined were the U.S. Food & Drug Administration's (FDA) Package Inserts (PIs), U.S. Centers for Disease Control and Prevention's (CDC) Vaccine Information Statements (VISs), and the European Medicines Agency's (EMA) Summary of Product Characteristics (SmPCs) and Package Leaflets (PLs). Prescribing information and patient information leaflet languages were subsequently organized into ten and seven categories, respectively. Comparison of FDA PIs to EMA SmPCs showed little harmonization between the two regions, and CDC VISs to EMA PLs revealed even less.