SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

AbstractA novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa)

Defensin-rich granules of human neutrophils: characterization of secretory properties

AbstractThe various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related α-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil α-defensins HNP 1–3. This allowed us to quantify the exocytosis of α-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of α-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release

Seigniorage and the International Payments

The α-defensins, human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed in promyelocytes and stored as mature HNPs in azurophil granules. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme that processes proHNPs nor the localization of processing has been identified. It has been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which are able to process recombinant proHNP into HNP in vitro. We investigated whether serine proteases are in fact responsible for processing of proHNP in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing in primary human bone marrow cells. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not enhance the proHNP processing capability. Finally, two patients suffering from Papillon-Lefèvre syndrome, who lack active neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s)

Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway

The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1alpha,25 dihydroxy-vitamin D3. Recent in vitro studies suggested that curcumin and poly-unsaturated fatty acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized that curcumin and PUFAs would induce expression of known VDR target genes in cells. In this study, we tested whether these compounds regulated two important VDR target genes - human cathelicidin antimicrobial peptide (CAMP) and 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1)- in human monocytic cell line U937, colon cancer cell line HT-29 and keratinocyte cell line HaCaT. We demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines, but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced CAMP promoter activity from a luciferase reporter construct lacking the VDR binding site and did not increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D receptor-independent manner. We conclude that PUFAs and curcumin do not function as ligands for the VDR.This is the authors' peer-reviewed accepted manuscript. The version of record is published and copyrighted by Elsevier and can be found at: http://www.elsevier.com/wps/find/journaldescription.cws_home/525013/description#descriptionKeywords: curcumin, vitamin D receptor, cathelicidin, poly-unsaturated fatty acid, innate immunit

Lipocalin-2 Functions as Inhibitor of Innate Resistance to Mycobacterium tuberculosis

Lipocalin-2 is a constituent of the neutrophil secondary granules and is expressed de novo by macrophages and epithelium in response to inflammation. Lipocalin-2 acts in a bacteriostatic fashion by binding iron-loaded siderophores required for bacterial growth. Mycobacterium tuberculosis (M.tb) produces siderophores that can be bound by lipocalin-2. The impact of lipocalin-2 in the innate immune response toward extracellular bacteria has been established whereas the effect on intracellular bacteria, such as M.tb, is less well-described. Here we show that lipocalin-2 surprisingly confers a growth advantage on M.tb in the early stages of infection (3 weeks post-challenge). Using mixed bone marrow chimeras, we demonstrate that lipocalin-2 derived from granulocytes, but not from epithelia and macrophages, leads to increased susceptibility to M.tb infection. In contrast, lipocalin-2 is not observed to promote mycobacterial growth at later stages of M.tb infection. We demonstrate co-localization of granulocytes and mycobacteria within the nascent granulomas at week 3 post-challenge, but not in the consolidated granulomas at week 5. We hypothesize that neutrophil-derived lipocalin-2 acts to supply a source of iron to M.tb in infected macrophages within the immature granuloma, thereby facilitating mycobacterial growth

Fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin with gemtuzumab ozogamicin improves event-free survival in younger patients with newly diagnosed aml and overall survival in patients with npm1 and flt3 mutations

Purpose To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. Patients and Methods One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). Results There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. Conclusion Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit

Neutrophils, from marrow to microbes

Neutrophils are produced in the bone marrow from stem cells that proliferate and differentiate to mature neutrophils fully equipped with an armory of granules. These contain proteins that enable the neutrophil to deliver lethal hits against microorganisms, but also to cause great tissue damage. Neutrophils circulate in the blood as dormant cells. At sites of infection, endothelial cells capture bypassing neutrophils and guide them through the endothelial cell lining whereby the neutrophils are activated and tuned for the subsequent interaction with microbes. Once in tissues, neutrophils kill microorganisms by microbicidal agents liberated from granules or generated by metabolic activation. As a final act, neutrophils can extrude stands of DNA with bactericidal proteins attached that act as extracellular traps for microorganisms