Platelet transfusions in a murine model of neonatal polymicrobial sepsis: Divergent effects on inflammation and mortality.

BACKGROUND: Platelet transfusions (PTxs) are often given to septic preterm neonates at high platelet count thresholds in an attempt to reduce bleeding risk. However, the largest randomized controlled trial (RCT) of neonatal transfusion thresholds found higher mortality and/or major bleeding in infants transfused at higher thresholds. Using a murine model, we investigated the effects of adult PTx on neonatal sepsis-induced mortality, systemic inflammation, and platelet consumption. STUDY DESIGN AND METHODS: Polymicrobial sepsis was induced via intraperitoneal injection of cecal slurry preparations (CS1, 2, 3) into P10 pups. Two hours after infection, pups were transfused with washed adult Green Flourescent Protein (GFP+) platelets or control. Weights, platelet counts, and GFP% were measured before 4 and 24 h post-infection. At 24 h, blood was collected for quantification of plasma cytokines. RESULTS: The CS batches varied in 24 h mortality (11%, 73%, and 30% in CS1, 2, and 3, respectively), due to differences in bacterial composition. PTx had differential effects on sepsis-induced mortality and systemic inflammatory cytokines, increasing both in mice infected with CS1 (low mortality) and decreasing both in mice infected with CS2 and 3. In a mathematical model of platelet kinetics, the consumption of transfused adult platelets was higher than that of endogenous neonatal platelets, regardless of CS batch. DISCUSSION: Our findings support the hypothesis that transfused adult platelets are consumed faster than endogenous neonatal platelets in sepsis and demonstrate that PTx can enhance or attenuate neonatal inflammation and mortality in a model of murine polymicrobial sepsis, depending on the composition of the inoculum and/or the severity of sepsis.

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features.

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic ß-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and ß-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the "brain-eating amoeba" Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria's spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers.

Development of gates to measure the immature platelet fraction in C57BL/6J mice using the Sysmex XN-V series multispecies hematology analyzer.

The immature platelet fraction (IPF) is a measure of newly released platelets, which has been used as a marker of platelet production in multiple human studies but is not widely available in multispecies analyzers. We developed gates to measure the IPF in diluted and undiluted murine blood samples on the Sysmex XN-1000V multispecies hematology analyzer. IPF gates were created using undiluted and diluted (1/10) blood samples obtained from adult and newborn (postnatal day 10, P10) C57BL/6J wild-type (WT) mice, and from 3 murine models of thrombocytopenia: c-MPL-/- mice, which lack the thrombopoietin receptor (hyporegenerative); antibody-mediated thrombocytopenia; and acute inflammation-induced thrombocytopenia. P10 mice were chosen because, at their size, we could consistently obtain (by terminal phlebotomy) the blood volume needed to run an undiluted sample. The undiluted blood IPF gate successfully differentiated between mechanisms of thrombocytopenia in both adult and P10 mice. For diluted samples, 2 IPF gates were generated: a thrombocytopenic (T) gate, which performed well in samples with platelet counts (PCs) <800 × 109/L in adult mice and <500 × 109/L in newborn mice, and a non-thrombocytopenic (NT) gate, which performed well in samples with PCs above these thresholds. PCs and IPFs measured in diluted blood using these gates agreed well with those measured in undiluted blood and had good reproducibility. These diluted gates allow for the accurate measurement of PCs and IPFs in small (10 µL) blood volumes, which can be obtained easily from adult and newborn mice as small as P1 to assess platelet production serially.