Stored RBC transfusions leads to the systemic inflammatory response syndrome in anemic murine neonates.

OBJECTIVE: RBC transfusions (RBCT) are life-saving treatment for premature and critically ill infants. However, the procedure has been associated with the development of systemic inflammatory response syndrome (SIRS) and potentially multiple organ dysfunction syndrome (MODS) in neonates. The present study aimed to investigate the mechanisms of RBCT-related SIRS in severely anemic murine neonates. METHODS: C57BL/6 (WT), TLR4-/- and myeloid-specific triggered myeloid receptor-1 (trem1)-/- mouse pups were studied in 4 groups (n = 6 each): (1) naïve controls, (2) transfused control, (3) anemic (hematocrit 20-24%) and (4) anemic with RBC transfused using our established murine model of phlebotomy-induced anemia (PIA) and RBC transfusion. Plasma was measured for quantifying inflammatory cytokines (IFN-γ, IL-1ß, TNF-α, IL-6, MIP-1α, MIP-1ß, MIP2 and LIX) using a Luminex assay. In vitro studies included (i) sensitization by exposing the cells to a low level of lipopolysaccharide (LPS; 500 ng/ml) and (ii) trem1-siRNA transfection with/without plasma supernatant from stored RBC to assess the acute inflammatory response through trem1 by qRT-PCR and immunoblotting. RESULTS: Anemic murine pups developed cytokine storm within 2 h of receiving stored RBCs, which increased until 6 h post-transfusion, as compared to non-anemic mice receiving stored RBCTs ("transfusion controls"), in a TLR4-independent fashion. Nonetheless, severely anemic pups had elevated circulating endotoxin levels, thereby sensitizing circulating monocytes to presynthesize proinflammatory cytokines (IFN-γ, IL-1ß, TNF-α, IL-6, MIP-1α, MIP-1ß, MIP2, LIX) and express trem1. Silencing trem1 expression in Raw264.7 cells mitigated both endotoxin-associated presynthesis of proinflammatory cytokines and the RBCT-induced release of inflammatory cytokines. Indeed, myeloid-specific trem1-/- murine pups had significantly reduced evidence of SIRS following RBCTs. CONCLUSION: Severe anemia-associated low-grade inflammation sensitizes monocytes to enhance the synthesis of proinflammatory cytokines and trem1. In this setting, RBCTs further activate these monocytes, thereby inducing SIRS. Inhibiting trem1 in myeloid cells, including monocytes, alleviates the inflammatory response associated with the combined effects of anemia and RBCTs in murine neonates.

Effect of RANKL on Lower Depressive Symptoms In Hemodialysis Patients.

Depression and osteoporosis are common diseases in dialysis patients. In addition, patients with osteoporosis are more susceptible to depression. Contrary to previous anti-osteoporosis agents, denosumab and romosozumab could be used in dialysis patients and have similar action mechanisms for blocking RANKL. RANKL causes bone resorption after binding RANKL, but binding with OPG leads to suppress of bone resorption. In recent mice study, inhibition of RANKL with denosumab improved depressive-like phenotype. Besides, it was found that OPG was associated with depression. Therefore, this study aimed to investigate the association of depressive symptoms with RANKL and OPG in hemodialysis patients. We conducted a cross-sectional study with a total of 172 hemodialysis patients. The participants were measured for plasma RANKL, OPG, MMP-2, and MMP-9 levels. Logistic regression analysis was performed to evaluate the effect of RANKL and OPG on the presence of depressive symptoms. The depressive symptoms were observed in 90 (52.3%) subjects. RANKL tertile 3 had negative association with BDI score (ß - 4.527, 95% CI - 8.310 to - 0.743) in univariate analysis, and this association persisted even after multivariate adjustments (ß - 5.603, 95% CI - 9.715 to -1.491) in linear regression. In logistic regression between RANKL tertiles and depressive symptoms, RANKL tertile 3 had significantly lower unadjusted OR (0.40, 95% CI 0.19-0.86), and multivariate-adjusted OR (0.31, 95% CI 0.12-0.82) for depressive symptoms. OPG was not significantly associated with depressive symptoms. Higher plasma RANKL concentrations were significantly associated with lower depressive symptoms in HD patients.Trial registration WHO registry, No. KCT0003281, date: January 12, 2017.

A Detailed Protocol for the Induction of Anemia and RBC Transfusion-associated Necrotizing Enterocolitis in Neonatal Mice.

Anemia is a common and serious health problem, nearly universally diagnosed in preterm infants, and is associated with increased morbidity and mortality worldwide. Red blood cell (RBC) transfusion is a lifesaving and mainstay therapy; however, it has critical adverse effects. One consequence is necrotizing enterocolitis (NEC), an inflammatory bowel necrosis disease in preterm infants. The murine model of phlebotomy-induced anemia and RBC transfusion-associated NEC enables a detailed study of the molecular mechanisms underlying these morbidities and the evaluation of potential new therapeutic strategies. This protocol describes a detailed procedure for obtaining murine pups with phlebotomy-induced anemia and delivering an RBC transfusion that develops NEC.

Hematological changes in neonatal mice with phlebotomy-induced anemia.

BACKGROUND: Anemia is a nearly universal diagnosis in preterm infants, caused by phlebotomy, and exacerbated by the underlying erythropoietic immaturity. Newborn infants are exposed to the unique stressor of fetal-to-neonatal transition, which requires significant adaptation ex utero. Accordingly, the preterm infant's response to anemia may alter the ability to confront underlying illness. This study utilized our preclinical mouse model of phlebotomy-induced anemia (PIA) to comprehensively investigate associated hematological changes. METHODS: C57BL/6 mice were subjected to timed phlebotomy between postnatal days 2--10 to induce severe anemia. Complete blood counts were determined by the Sysmex XT-2000iV analyzer. RESULTS: Anemic pups showed a gradual reduction of RBC and hemoglobin (Hb) and increased reticulocyte (RET) counts and red cell distribution width (RDW), however, with reduced RET-Hb from postnatal day (P) of 4 onwards. Elevated levels of high fluorescent RET and immature reticulocyte fraction (IRF) were noted in anemic mouse pups, but low and medium fluorescent RET were reduced. Also, the reduction of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were noted in anemic pups. No changes were seen in lymphocytes, but monocytes and neutrophils were significantly elevated from P4-P6. CONCLUSIONS: PIA in mouse pups is associated with hematological changes that may be exacerbating factors in neonatal diseases. IMPACT: Anemia is common and often severe in premature infants. Investigation of hematological parameters in settings of preclinical anemia may be an index of therapeutic strategies. Preclinical model evaluating the effects of neonatal anemia on the remainder of complete blood count. Detailed time kinetic phlebotomy-induced anemic mice enable us to study the impact on developmental delays in erythropoiesis and possible strategic intervention. Hematological effects of severe anemia in mice might provide insight on how best to investigate anemia in preterm infants.

Severe neonatal anemia increases intestinal permeability by disrupting epithelial adherens junctions.

Anemia is a frequent diagnosis in critically ill infants, but the clinical implications of severe anemia in these patients remain unclear. In this study, we examined preweaned mice to investigate the effects of severe anemia during early infancy on gut mucosal permeability. C57BL/6 mice were subjected to timed phlebotomy between postnatal days (P) 2-10 to induce severe anemia (hematocrits 20%-24%), and intestinal permeability was tracked longitudinally between P10 and P20 as intestine-to-plasma translocation of enteral macromolecules and bacterial translocation. Epithelial junctions were evaluated by electron microscopy, polymerase chain reactions, immunohistochemistry, and/or enzyme immunoassays on intestinal tissues, Caco-2 intestinal epithelial-like cells, and colonic organoids. Preweaned mouse pups showed an age-related susceptibility to severe anemia, with increased intestinal permeability to enteral macromolecules (dextran, ovalbumin, ß-lactoglobulin) and luminal bacteria. Electron micrographs showed increased paracellular permeability and ultrastructural abnormalities of the adherens junctions. These findings were explained by the loss of E-cadherin in epithelial cells, which was caused by destabilization of the E-cadherin (Cdh1) mRNA because of microRNA let-7e-5p binding to the 3'-untranslated region. Severe anemia resulted in a disproportionate and persistent increase in intestinal permeability in preweaned mice because of the disruption of epithelial adherens junctions. These changes are mediated via microRNA let-7e-mediated depletion of Cdh1 mRNA.NEW & NOTEWORTHY This research article shows that newborn infants with severe anemia show an age-related susceptibility to developing increased intestinal permeability to ingested macromolecules. This abnormal permeability develops because of abnormalities in intestinal epithelial junctions caused by a deficiency of the molecule E-cadherin in epithelial cells. The deficiency of E-cadherin is caused by destabilization of its mRNA precursor because of increased expression and binding of another molecule, the microRNA let-7e-5p, to the E-cadherin mRNA.

Lyophilization of human amniotic fluid is feasible without affecting biological activity.

BACKGROUND: Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs. METHODS: We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74. RESULTS: After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p < 0.001) and decreased LPS-induced IL-8 production (p < 0.01). CONCLUSIONS: Lyophilization and filtration of hAF is feasible with partial loss of TFs but maintains and even improves bioavailability of TFs measured by proliferation and LPS-induced IL-8 production by FHs74.