Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans

Accuracy of Risk Estimation for Surgeons Versus Risk Calculators in Emergency General Surgery

IntroductionSurgical risk calculators have expanded in both number and sophistication of their predictive approach. These calculators are gaining popularity as validated tools to help surgeons estimate mortality and complications following emergency general surgery (EGS). However, the accuracy of risk estimates generated by these calculators compared to risk estimation by practicing surgeons has not been explored.MethodsAcute care surgeons at a quaternary care center prospectively estimated 30-d mortality and complications for adult EGS patients (2019-2021). Surgeon predictions were compared to Predictive OpTimal Trees in Emergency Surgery Risk (POTTER) and NSQIP estimates. Observed-to-expected (O:E) ratios of median aggregate estimates were calculated. C-statistics for surgeon and calculator estimations were utilized to quantify predictive accuracy.ResultsAmong 150 patients (median 61 y, 45% male), 30-d mortality was 15% (n = 23). Observed rates of prolonged mechanical ventilation and acute renal failures were 30% and 10%, respectively. Overall, surgeon predictions were similar to risk calculator estimates for mortality (c-statistics 0.843 [surgeon] versus 0.848 [POTTER] and 0.815 [NSQIP]) and need for prolonged ventilation (c-statistics 0.801 versus 0.722 and 0.689, respectively). Surgeons tended to overestimate complication risks. Surgeon experience was not significantly associated with mortality prediction in an adjusted model.ConclusionsAcute care surgeons at a quaternary care center predicted postoperative mortality and complications with similar discrimination when compared to surgical risk calculators. Surgeon expertise should be utilized in conjunction with risk calculators when counseling EGS patients regarding anticipated postoperative outcomes. Surgeons should be cognizant of patterns in overestimation or underestimation of complications

LRG1 predominantly localizes to the secondary granule compartment of neutrophils.

<p><b>(A)</b> Neutrophils were isolated from the peripheral blood of healthy donors as described, lysed by nitrogen cavitation, and the subcellular components separated by Percoll density centrifugation. The fractions are numbered 1 through 30, with fraction 1 corresponding to the most dense fraction and fraction 30 corresponding to the least dense fraction. Fractions were analyzed by ELISA for the presence of LRG1, MPO (primary granule marker), LF (secondary granule marker), and MMP9 (tertiary granule marker), and the results plotted as a function of dentisty. <b>(B)</b> Immunoblot analyses of fractions 1 through 21 from the same experiment presented in panel A, showing peak concentrations for MPO, LF, and MMP9 in fractions 2, 10, and 17, respectively, identical to the fractions containing the highest concentration of the indicated proteins as detected by ELISA shown in panel A. The peak concentration of LRG1 is detected in fraction 10, which corresponds to the peroxidase-negative LF-containing secondary granule compartment, consitant with the ELISA data presented in Panel A. The data shown are representative of 5 independent experiments.</p

LRG1 modifies the effects of TGFβ on myeloid and hematopoietic progenitor cell growth.

<p><b>(A)</b> HL-60 cell proliferation in the presence of increasing concentrations of TGFβ alone (blue diamonds) or TGFβ plus LRG1 (800 ng/mL, red squares) was assessed using the XTT assay. A dose-response reduction in cell proliferation as measured by the decreasing A490-A650 is observed with increasing concentrations of TGFβ that is mitigated by the addition of LRG1. The Student's t-test was used to determine the significance of the differences between control and LRG1 treated samples († p < 0.05) (n = 5). <b>(B)</b> Purified bone marrow-derived CD34+ cells were cultured in serum-free semi-solid media in the presence of TGFβ (10 ng/mL) with or without LRG1 at the indicated concentrations, and colony numbers scored. An increase in colony numbers relative to TGFβ1 treatment alone (0) is seen with increasing concentrations of LRG1. The Student's t-test was used to determine the significance of the differences between control and LRG1 treated samples († p < 0.05) (n = 3). <b>(C)</b> The effects of TGFβ alone (left panel) and TGFβ plus LRG1 (right panel) on colony formation and colony composition were analyzed. <i>Upper two images in each panel</i>. Representative light microscopy images of methocult plates on which bone marrow-derived CD34+ cells were cultured in the presence of TGFβ alone (left) or TGFβ with LRG1 (right). <i>Lower two images in each panel</i>. Wright-Giemsa staining of cells plucked from individual colonies from the methocult plates in the upper images is shown. Cells were grown as described in (B) in the presence of TGFβ (10 ng/mL) with or without LRG1 (0.625 ug/mL). LRG1 mitigates the inhibitory effect of TGFβ on the growth and size of colony-forming units-granulocyte monocyte (CFU-GM). The data shown are representative of five independent experiments, each with a different normal donor.</p

Multi-Omic Admission-Based Prognostic Biomarkers Identified by Machine Learning Algorithms Predict Patient Recovery and 30-Day Survival in Trauma Patients

Admission-based circulating biomarkers for the prediction of outcomes in trauma patients could be useful for clinical decision support. It is unknown which molecular classes of biomolecules can contribute biomarkers to predictive modeling. Here, we analyzed a large multi-omic database of over 8500 markers (proteomics, metabolomics, and lipidomics) to identify prognostic biomarkers in the circulating compartment for adverse outcomes, including mortality and slow recovery, in severely injured trauma patients. Admission plasma samples from patients (n = 129) enrolled in the Prehospital Air Medical Plasma (PAMPer) trial were analyzed using mass spectrometry (metabolomics and lipidomics) and aptamer-based (proteomics) assays. Biomarkers were selected via Least Absolute Shrinkage and Selection Operator (LASSO) regression modeling and machine learning analysis. A combination of five proteins from the proteomic layer was best at discriminating resolvers from non-resolvers from critical illness with an Area Under the Receiver Operating Characteristic curve (AUC) of 0.74, while 26 multi-omic features predicted 30-day survival with an AUC of 0.77. Patients with traumatic brain injury as part of their injury complex had a unique subset of features that predicted 30-day survival. Our findings indicate that multi-omic analyses can identify novel admission-based prognostic biomarkers for outcomes in trauma patients. Unique biomarker discovery also has the potential to provide biologic insights

LRG1 is expressed during neutrophilic granulocyte differentiation and stored in lactoferrin (LF)-containing granules.

<p><b>(A)</b> HL-60 cells were induced to differentiate with ATRA (1 uM) for 6 days, and soluble proteins extracted from cells harvested at days 0, 1, 3, and 6. Samples were subjected to immunoblot analysis to detect the presence of LRG1. Pure LRG1 in lane 1 is LRG1 purified from human serum included as a positive control. Proteins isolated from unstimulated human neutrophils (PMN lysate) are shown in the last lane. (<b>B)</b> Confocal immunofluorescence microscopy of purified human neutrophils incubated overnight with mouse anti-LRG1 (Abnova H00116844-M01, 1:100 dilution) and goat anti-LF (Santa Cruz sc-14431, 1:250 dilution), followed by donkey anti-mouse Alexafluor 488 (green, 1:500 dilution) and donkey anti-goat Alexafluor 594 (red, 1:2000 dilution) secondary antibodies. DAPI was used to stain nuclei (blue). LRG1 is visualized as green and LF as red. The yellow/orange color in the overlay panel is the result of green and red signals merging, indicating co-localization of LRG1 (green) with LF (red). Focal areas of non-merged green and red signals can also be seen, suggesting that LRG1 may also localize to non-LF-containing compartments. The data shown are representative of 3 independent experiments.</p

Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1