A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs.

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro.

A pro-tumorigenic function of S100A8/A9 in carcinogen-induced hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is a heterogeneous disease, driven by different risk factors and presenting diverse clinicopathological features and outcomes. Epidemiological and experimental data indicate that the damage-associated molecular pattern molecules S100A8 and S100A9, forming a heterodimer called calprotectin, might be critically involved in HCC development. However, deletion of S100a9 in an inflammation- and cirrhosis-driven mouse model did not show any impairment in liver tumorigenesis, most likely due to functional compensation by other inflammatory cytokines.

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction.

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area.

Chronic liver inflammation and hepatocellular carcinogenesis are independent of S100A9.

The S100A8/A9 heterodimer (calprotectin) acts as a danger signal when secreted into the extracellular space during inflammation and tissue damage. It promotes proinflammatory responses and drives tumor development in different models of inflammation-driven carcinogenesis. S100A8/A9 is strongly expressed in several human tumors, including hepatocellular carcinoma (HCC).

A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia.

The S100A8/A9 heterodimer is abundantly expressed by myeloid cells, especially neutrophils, but its mechanism of action is only partially determined. In this study we investigated S100A8/A9 involvement in the host response to Streptococcus pneumoniae infection making use of S100a9(-/-) mice that lack heterodimer expression in myeloid cells. S100a9(-/-) mice that were infected intranasally with pneumococci rapidly succumbed, with 80% mortality after 48 h, whereas the majority of wild-type mice recovered.

Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis.

Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14⁻/⁻ mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14⁻/⁻ mice.

S100A9 has a protective role in inflammation-induced skin carcinogenesis.

The S100A8/A9 heterodimer is expressed by myeloid cells where its function has been extensively investigated. Immune cell S100A8/A9 promotes proinflammatory effects, and its absence is often associated with lack of leukocyte recruitment resulting in protection in terms of disease progression. S100A8/A9 is also expressed by certain epithelia, either constitutively as in mucosal epithelia or following stimulation as in skin keratinocytes.

A new leukocyte hyperadhesion syndrome of delayed cord separation, skin infection, and nephrosis.

Leukocyte adhesion deficiency (LAD) I is a well-described genetic disorder in which leukocytes are unable to migrate to sites of inflammation due to mutations in the ITGB2 gene coding for the β subunit of β2 (CD18) leukocyte integrins. The classic symptoms of the disease present in the newborn period as failure of separation of the umbilical cord and recurrent bacterial infections, which continue throughout life. We report on a patient with these clinical manifestations but with normal ITGB2 gene sequencing excluding LAD-I, normal carbohydrate-deficient transferrin testing excluding LAD-II, and normal platelet function excluding LAD-III.

S100A8-S100A9 protein complex mediates psoriasis by regulating the expression of complement factor C3.

Psoriasis is a common heterogeneous inflammatory skin disease with a complex pathophysiology and limited treatment options. Here we performed proteomic analyses of human psoriatic epidermis and found S100A8-S100A9, also called calprotectin, as the most upregulated proteins, followed by the complement component C3. Both S100A8-S100A9 and C3 are specifically expressed in lesional psoriatic skin.

SHARPIN regulates uropod detachment in migrating lymphocytes.

SHARPIN-deficient mice display a multiorgan chronic inflammatory phenotype suggestive of altered leukocyte migration. We therefore studied the role of SHARPIN in lymphocyte adhesion, polarization, and migration. We found that SHARPIN localizes to the trailing edges (uropods) of both mouse and human chemokine-activated lymphocytes migrating on intercellular adhesion molecule-1 (ICAM-1), which is one of the major endothelial ligands for migrating leukocytes.

Novel automated tracking analysis of particles subjected to shear flow: kindlin-3 role in B cells.

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images.

Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.

Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation.

The kindlin 3 pleckstrin homology domain has an essential role in lymphocyte function-associated antigen 1 (LFA-1) integrin-mediated B cell adhesion and migration.

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides.

A role for LFA-1 in delaying T-lymphocyte egress from lymph nodes.

Lymphocytes use the integrin leukocyte function-associated antigen-1 (LFA-1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA-1 dependent. We show that LFA-1 mediates prolonged LN residence as LFA-1(-/-) CD4 T cells have significantly decreased dwell times compared with LFA-1(+/+) T cells, a distinction lost in hosts lacking the major LFA-1 ligand ICAM-1. Intra-vital two-photon microscopy revealed that LFA-1(+/+) and LFA-1(-/-) T cells reacted differently when probing the ICAM-1-expressing lymphatic network.

T lymphocytes orient against the direction of fluid flow during LFA-1-mediated migration.

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.

Chronic lymphocytic leukemia cells induce defective LFA-1-directed T-cell motility by altering Rho GTPase signaling that is reversible with lenalidomide.

T lymphocytes have an essential role in adaptive immunity and rely on the activation of integrin lymphocyte function-associated antigen-1 (LFA-1) to mediate cell arrest and migration. In cancer, malignant cells modify the immune microenvironment to block effective host antitumor responses. We show for the first time that CD4 and CD8 T cells from patients with chronic lymphocytic leukemia (CLL) exhibit globally impaired LFA-1-mediated migration and that this defect is mediated by direct tumor cell contact.

A role for Rap2 in recycling the extended conformation of LFA-1 during T cell migration.

T lymphocytes make use of their major integrin LFA-1 to migrate on surfaces that express ICAM-1 such as blood vessels and inflamed tissue sites. How the adhesions are turned over in order to supply traction for this migration has not been extensively investigated. By following the fate of biotinylated membrane LFA-1 on T lymphocytes, we show in this study that LFA-1 internalization and re-exposure on the plasma membrane are linked to migration.

A CXCL1 paracrine network links cancer chemoresistance and metastasis.

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites.

The Gαq/11 proteins contribute to T lymphocyte migration by promoting turnover of integrin LFA-1 through recycling.

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration.

Loss of myeloid related protein-8/14 exacerbates cardiac allograft rejection.

Background: The calcium-binding proteins myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) form MRP-8/14 heterodimers (S100A8/A9, calprotectin) that regulate myeloid cell function and inflammatory responses and serve as early serum markers for monitoring acute allograft rejection. Despite functioning as a proinflammatory mediator, the pathophysiological role of MRP-8/14 complexes in cardiovascular disease is incompletely defined. This study investigated the role of MRP-8/14 in cardiac allograft rejection using MRP-14(-/-) mice that lack MRP-8/14 complexes.

The insider's guide to leukocyte integrin signalling and function.

The activation of leukocyte integrins through diverse receptors results in transformation of the integrin from a bent, resting form to an extended conformation, which has at least two states of ligand-binding activity. This highly regulated activation process is essential for T cell migration and the formation of an immunological synapse. The signalling events that drive integrin activation are complex.

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice.

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture.

The integrins Mac-1 and alpha4beta1 perform crucial roles in neutrophil and T cell recruitment to lungs during Streptococcus pneumoniae infection.

Neutrophils and T cells play an important role in host protection against pulmonary infection caused by Streptococcus pneumoniae. However, the role of the integrins in recruitment of these cells to infected lungs is not well understood. In this study we used the twin approaches of mAb blockade and gene-deficient mice to investigate the relative impact of specific integrins on cellular recruitment and bacterial loads following pneumococcal infection.

G-CSF-mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands.

Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice.