Placental HTRA1 cleaves alpha-1-antitrypsin to generate a NET-Inhibitory Peptide

Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (PMNs) fail to form NETs due to circulating NET-Inhibitory Peptides (NIPs) -cleavage fragments of alpha-1-antitrypsin (A1AT). However, how fetal and neonatal blood NIPs are generated remains unknown. The placenta expresses High-Temperature Requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1-knock out mice (HTRA1-/-). We found that umbilical cord blood plasma has elevated HTRA1 levels compared to adult plasma, and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of similar molecular weight to previously identified NIPs that block NET formation by adult neutrophils. We demonstrated that neonatal mouse pup plasma contains A1AT fragments which inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. LPS-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation following birth. However, plasma from HTRA1-/- pups had no detectable NIPs and PMNs from HTRA1-/- pups became NET competent earlier after birth compared to HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential “developmental hemostatic mismatch risk” associated with transfusions containing plasma exosomes from adults.

Non-Targeted Metabolomics Signature in the Plasma and Bone Marrow of Patients with Long Bone Injuries

Background: The contribution of long bone injury and reaming to the inflammatory response of trauma is unknown. Introduction: This study evaluated whether metabolomics can be used to (1) reveal differences in the plasma from long bone injury trauma patients before and after reaming and (2) distinguish healthy adult plasma from that of trauma patients. Methods: Prospective cohort study with enrollment from February 17, 2017 to December 5, 2017 included 15 patients with long bone injuries and 20 healthy adults. Patients with femoral or tibial fractures scheduled to undergo intramedullary nailing were identified at the Medical Center of the Rockies, (Loveland, Co), and Memorial Hospital, (Colorado Springs, CO). Pre-and post-reaming plasma and bone marrow from fifteen patients with femoral and tibial fractures and 20 heathy adult plasma were analyzed by ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). Results: Trauma patients had 1259 plasma metabolites and healthy adults had 1272 plasma metabolites detected. Fifty percent (657 metabolites) were common between the bone marrow and plasma profiles, and 304 metabolites showed statistical significance for differential abundance between pre- and post-reaming (P<0.05). Post-ream lipids, fatty acids and ceramides were 1.09-1.46-fold increased and diacylglycerols were 0.73-0.82-fold decreased compared to the pre-ream patient control. Post-ream tryptophan metabolites were decreased 0.84-fold, whereas cysteine metabolites were elevated 1.42-fold. Metabolite signals associated with bone matrix remodeling, stress and inflammation were modulated in all patients. Conclusion: Plasma metabolite signatures changed in long bone fracture patients pre- and post-reaming showing distinct profiles from healthy adults without trauma injury. Metabolite signatures of long bone trauma patients have an inflammatory response reflective of healing cascades and merits additional testing for markers of individualized responses to injury.

Measuring Antibody Avidity to Plasmodium Falciparum Merozoite Antigens Using a Multiplex Immunoassay Approach

Background: Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration.Methods: The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to i) identify the assay with the widest range of AI (discriminatory power), ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and iii) evaluate assay repeatability. Results: Overall, 4M GdHCl and 8M urea were weaker chaotropes than 3M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 one-year old infants in Cameroon showed that 2.1M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8M ± 0.23M (infants) released 50% of bound Ab from the merozoite antigens. Conclusions: An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

Measuring Antibody Avidity to Plasmodium Falciparum Merozoite Antigens Using a Multiplex Immunoassay Approach

Background: Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration.Methods: The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to i) identify the assay with the widest range of AI (discriminatory power), ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and iii) evaluate assay repeatability. Results: Overall, 4M GdHCl and 8M urea were weaker chaotropes than 3M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 one-year old infants in Cameroon showed that 2.1M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8M ± 0.23M (infants) released 50% of bound Ab from the merozoite antigens. Conclusions. An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

Apixaban pharmacodynamic activity in umbilical cord, paediatric, and adult plasma

SummaryThe objective was to characterise apixaban pharmacodynamic (PD) activity in umbilical cord (UC), paediatric, and adult plasma. Plasma was obtained from blood samples from six UC donors, 70 paediatric (neonates [birth–≤1 month], infants [>1–≤6 months], toddlers [>6 months–≤2 years], young children [>2–≤6 years], children [>6–≤12 years], adolescents [>12–≤18 years]), and six adult (19–45 years) subjects. Plasma spiked with apixaban 0 (baseline), 30, or 110 ng/ml was analysed for anti-factor Xa activity, factor X levels, prothrombin time (PT), and modified PT (mPT). Apixaban had similar concentration-related effects on anti-factor Xa activity across groups (30 ng/ml: 0.223–0.295 IU/ml; 110 ng/ml: 1.212–1.474 IU/ml). Endogenous baseline factor X levels were 43%–68% lower in plasma from UC and subjects ≤6 months versus adults. Factor Xa inhibition (percentage change from baseline in apparent factor X levels) was similar for both apixaban concentrations across groups, except UC, neonate, and infant groups, which showed greater inhibition vs adults for apixaban 110 ng/ml. Baseline PT and mPT were similar across groups. Apixaban had no effect on PT at the concentrations tested. Apixaban 110 ng/ml prolonged mPT similarly across groups (44.4–53.2 s to 64.5–70.0 s); no prolongation was found with apixaban 30 ng/ml. Apixaban demonstrated consistent concentration-related effects on other PD endpoints in plasma samples from all age groups, except factor Xa inhibition.Supplementary Material to this article is available at www.thrombosis-online.com.

Proteomic Analysis of the Ontogenetic Variability in Plasma Composition of Juvenile and Adult Bothrops jararaca Snakes

The ontogenetic variability in venom composition of some snake genera, including Bothrops, as well as the biological implications of such variability and the search of new molecules that can neutralize the toxic components of these venoms have been the subject of many studies. Thus, considering the resistance of Bothrops jararaca to the toxic action of its own venom and the ontogenetic variability in venom composition described in this species, a comparative study of the plasma composition of juvenile and adult B. jararaca snakes was performed through a proteomic approach based on 2D electrophoresis and mass spectrometry, which allowed the identification of proteins that might be present at different levels during ontogenetic development. Among the proteins identified by mass spectrometry, antihemorrhagic factor Bj46a was found only in adult plasma. Moreover, two spots identified as phospholipase A2 inhibitors were significantly increased in juvenile plasma, which can be related to the higher catalytic PLA2 activity shown by juvenile venom in comparison to that of adult snakes. This work shows the ontogenetic variability of B. jararaca plasma, and that these changes can be related to the ontogenetic variability described in its venom.