Leukocyte adhesion deficiencies.

During inflammation, leukocytes play a key role in maintaining tissue homeostasis through elimination of pathogens and removal of damaged tissue. Leukocytes migrate to the site of inflammation by crawling over and through the blood vessel wall, into the tissue. Leukocyte adhesion deficiencies (ie, LAD-I, -II, and LAD-I/variant, the latter also known as LAD-III) are caused by defects in the adhesion of leukocytes to the vessel wall, resulting in severe recurrent nonpussing infections and neutrophilia, often preceded by delayed separation of the umbilical cord. Although dependent on the genetic defect, hematopoietic stem cell transplantation is often the only curative treatment.

A novel flow cytometry-based platelet aggregation assay.

The main function of platelets is to maintain normal hemostasis. Inefficient platelet production and/or defective platelet function results in bleeding disorders resulting from a wide range of genetic traits and acquired pathologies. Several platelet function tests have been developed for use in the clinic and in experimental animal models. In particular, platelet aggregation is routinely measured in an aggregometer, which requires normal platelet counts and significant blood sample volumes. For this reason, the analysis of thrombocytopenic patients, infants, and animal models is problematic. We have developed a novel flow cytometry test of platelet aggregation, in which 10- to 25-fold lower platelet counts or sample volumes can be used, either of platelet-rich plasma or whole blood from human subjects or mice. This setup can be applied to test in small assay volumes the influence of a variety of stimuli, drugs, and plasma factors, such as antibodies, on platelet aggregation. The presented principle stands as a novel promising tool, which allows analysis of platelet aggregation in thrombocytopenic patients or infants, and facilitates studies in platelets obtained from experimental animal models without the need of special devices but a flow cytometer.

Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin ß1 and ß3 in platelet adhesion to collagen.

Patients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbß3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2ß1) instead of integrin αIIbß3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.

Hematologically important mutations: leukocyte adhesion deficiency (first update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, mutations are found in ITGB2, the gene that encodes the ß subunit of the ß(2) integrins. This syndrome is characterized directly after birth by delayed separation of the umbilical cord. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Le(a) and Le(b) blood group antigens. Finally, in LAD-III (also called LAD-I/variant) the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells that is involved in the regulation of ß integrin conformation.

LAD-1/variant syndrome is caused by mutations in FERMT3.

Leukocyte adhesion deficiency-1/variant (LAD1v) syndrome presents early in life and manifests by infections without pus formation in the presence of a leukocytosis combined with a Glanzmann-type bleeding disorder, resulting from a hematopoietic defect in integrin activation. In 7 consanguineous families, we previously established that this defect was not the result of defective Rap1 activation, as proposed by other investigators. In search of the genetic defect, we carried out homozygosity mapping in 3 of these patients, and a 13-Mb region on chromosome 11 was identified. All 7 LAD1v families share the same haplotype, in which 3 disease-associated sequence variants were identified: a putative splice site mutation in CALDAGGEF1 (encoding an exchange factor for Rap1), an intronic 1.8-kb deletion in NRXN2, and a premature stop codon (p.Arg509X) in FERMT3. Two other LAD1v patients were found to carry different stop codons in FERMT3 (p.Arg573X and p.Trp229X) and lacked the CALDAGGEF1 and NRXN2 mutations, providing convincing evidence that FERMT3 is the gene responsible for LAD1v. FERMT3 encodes kindlin-3 in hematopoietic cells, a protein present together with integrins in focal adhesions. Kindlin-3 protein expression was undetectable in the leukocytes and platelets of all patients tested. These results indicate that the LAD1v syndrome is caused by truncating mutations in FERMT3.

Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures.

Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll-carotenoid protein complex known as IsiA or CP43'. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.