Iris M. De Cuyper

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.

Analysis of the effect of highly active antiretroviral therapy during acute HIV-1 infection on HIV-specific CD4 T cell functions

Background:It has been reported that antiretroviral therapy (HAART) during acute HIV-1 infection may rescue HIV-1-specific CD4 T cell responses. Objective:To determine the duration of this preserved response by investigating the long-term effects of HAART during acute infection on HIV-specific CD4 T cell function related to possible immune control during subsequent therapy interruption. Methods:A longitudinal analysis followed HIV-specific CD4 T cell reactivity in 17 individuals with well-documented acute HIV-1 infection where five out of 11 HAART-treated patients stopped therapy and six were untreated. Peripheral blood mononuclear cells were stimulated with overlapping peptide pools derived from Gag and Nef. Production of interferon-γ (IFN-γ) and interleukin-2 (IL-2) by CD4 T cells was analysed together with proliferative responses. Results:Absolute numbers, but not percentages, of Gag-specific IFN-γ-, IL-2- or IFN-γ/IL-2-producing CD4 T cells were increased in treated compared with untreated individuals up to 2 years after seroconversion. HAART during acute HIV-1 infection was associated with lower viral load but did not result in increased proliferation of HIV-specific CD4 T cells. One out of five individuals who discontinued therapy showed evidence for immune control. However, patients who failed to control viraemia also had measurable proliferative HIV-specific CD4 T cell responses and preserved numbers of cytokine-producing CD4 T cells. Conclusions:Early HAART during acute HIV-1 infection resulted in higher numbers of HIV-specific IFN-γ- and IL-2-producing CD4 T cells, but this preservation in four out of five patients was not associated with control of viraemia upon treatment interruption.

UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with UV-C light (1500 J/m(2)) caused platelet aggregation, which was dependent on integrin alphaIIbbeta3 activation (GPIIb/IIIa). This activation occurred despite treatment with several signal transduction inhibitors known to block platelet activation. UV-C also induced activation of recombinant alphaIIbbeta3 in Chinese hamster ovary (CHO) cells, an environment in which physiologic agonists fail to activate. Activation of alphaIIbbeta3 requires talin binding to the beta3 tail, yet alphaIIbbeta3-Delta724 (lacking the talin binding site) was activated by UV-C irradiation, excluding a requirement for talin binding. The UV-C effect appears to be general in that beta(1) and beta(2) integrins are also activated by UV-C. To explain these findings, we investigated the possibility of UV-C-induced photolysis of disulfide bonds, in analogy with the activating effect of reducing agents on integrins. Indeed, UV-C induced a marked increase in free thiol groups in platelet surface proteins including alphaIIbbeta3. Thus, UV-C appears to activate alphaIIbbeta3 not by affecting intracellular signal transduction, but by reduction of disulfide bonds regulating integrin conformation.

Abundance of early functional HIV-specific CD8+ T cells does not predict AIDS-free survival time

Background T-cell immunity is thought to play an important role in controlling HIV infection, and is a main target for HIV vaccine development. HIV-specific central memory CD8+ and CD4+ T cells producing IFNγ and IL-2 have been associated with control of viremia and are therefore hypothesized to be truly protective and determine subsequent clinical outcome. However, the cause-effect relationship between HIV-specific cellular immunity and disease progression is unknown. We investigated in a large prospective cohort study involving 96 individuals of the Amsterdam Cohort Studies with a known date of seroconversion whether the presence of cytokine-producing HIV-specific CD8+ T cells early in infection was associated with AIDS-free survival time. Methods and Findings The number and percentage of IFNγ and IL-2 producing CD8+ T cells was measured after in vitro stimulation with an overlapping Gag-peptide pool in T cells sampled approximately one year after seroconversion. Kaplan-Meier survival analysis and Cox proportional hazard models showed that frequencies of cytokine-producing Gag-specific CD8+ T cells (IFNγ, IL-2 or both) shortly after seroconversion were neither associated with time to AIDS nor with the rate of CD4+ T-cell decline. Conclusions These data show that high numbers of functional HIV-specific CD8+ T cells can be found early in HIV infection, irrespective of subsequent clinical outcome. The fact that both progressors and long-term non-progressors have abundant T cell immunity of the specificity associated with low viral load shortly after seroconversion suggests that the more rapid loss of T cell immunity observed in progressors may be a consequence rather than a cause of disease progression.

High responsiveness of HLA‐B57‐restricted Gag‐specific CD8+ T cells in vitro may contribute to the protective effect of HLA‐B57 in HIV‐infection

HLA‐B57 has been shown to be associated with long‐term asymptomatic HIV‐1 infection. To investigate the biological mechanism by which the HLA‐B57 allele could protect from HIV‐1 disease, we studied both the number of CD8+ T cells as well as CD8+ T cell responsiveness directed to different HIV‐1 Gag peptides presented by HLA‐A2, ‐B8 or ‐B57. T cells specific for the HLA‐B57 peptide KAFSPEVIPMF responded more readily and to a higher extend to antigenic stimulation in vitro than T cells specific for the HLA‐A2 peptide SLYNTVATL or the HLA‐B8 peptide EIYKRWII. This phenomenon was reproducible with T cells from individuals expressing HLA‐B57 in combination with one or both of the other alleles and was persistent during long‐term follow‐up. Lower reactivity of A2‐ and B8‐restricted T cells was not explained by mutations in the B8‐ or A2‐restricted Gag‐peptides. Moreover, no correlation between peptide mutation frequency and IFN‐γ production by the corresponding Gag‐specific T cells was observed. In conclusion, functional differences were observed between T cells specific for HIV epitopes derived from the same protein presented by different HLA molecules. B57‐restricted KAFSPEVIPMF‐specific CD8+ T cells have relatively high responsiveness, which could contribute to the protective effect of HLA‐B57 in HIV infection.

Pathogen reduction treatment using riboflavin and ultraviolet light impairs platelet reactivity toward specific agonists in vitro.

Recent studies showed that Mirasol pathogen reduction treatment (PRT) leads to increased P‐selectin expression and increased oxygen and glucose consumption in resting platelets (PLTs). This study investigates the effect of PRT on PLT activation.

Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 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 αIIbβ3), 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 α2β1) instead of integrin αIIbβ3, 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.

Reconstitution of EBV Latent but Not Lytic Antigen-Specific CD4+ and CD8+ T Cells after HIV Treatment with Highly Active Antiretroviral Therapy

The incidence of (EBV-related) malignancies in HIV-infected subjects has declined since the introduction of highly active antiretroviral therapy (HAART). To investigate the effect of HAART on EBV infection, we performed a longitudinal analysis of the T cell response to both a latent and a lytic Ag and EBV viral load in 10 subjects from early in HIV infection up to 5 years after HAART. All individuals responded to HAART by a decline in HIV viral load, a restoration of total CD4+ T cell numbers, and a decline in T cell immune activation. Despite this, EBV load remained unaltered, even after 5 years of therapy, although a decline in both CD4+ and CD8+ T cells specific for the lytic EBV protein BZLF1 suggested a decreased EBV reactivation rate. In contrast, latent EBV Ag EBNA1-specific CD4+ and CD8+ T cell responses were restored after 5 years of treatment to levels comparable to healthy individuals. In two individuals who were treated by HAART late during HIV progression, a lymphoma developed shortly after initiation of HAART, despite restoration of EBV-specific CD4+ and CD8+ T cells. In conclusion, long-term HAART does not alter the EBV DNA load, but does lead to a restoration of EBNA1-specific T cell responses, which might allow better control of EBV-infected cells when applied early enough during HIV infection.

Noninvasive measurement of pH in platelet concentrates with a fiber optic fluorescence detector

BACKGROUND: Stored platelets (PLTs) are metabolically active, resulting in a decrease of pH during storage. The pH of PLT concentrates (PCs) is recognized as a measure of quality, and pH limits are set by regulatory bodies. A pH sensor was built into a PLT storage container, and the feasibility of testing pH using a noninvasive fluorescent measurement method was evaluated.

Erythropoietic Defect Associated with Reduced Cell Proliferation in Mice Lacking the 26S Proteasome Shuttling Factor Rad23b

ABSTRACT Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely due to the redundant function of the Rad23b paralogue, Rad23a. However, Rad23b-null midgestation embryos are anemic, and most embryos die before birth. Using an unbiased proteomics approach, we found that the majority of Rad23b-interacting partners are associated with the ubiquitin-proteasome system (UPS). We tested the requirement for Rad23b-dependent UPS activity in cellular proliferation and more specifically in the process of erythropoiesis. In cultured fibroblasts derived from embryos lacking Rad23b, proliferation rates were reduced. In fetal livers of Rad23b-null embryos, we observed reduced proliferation, accumulation of early erythroid progenitors, and a block during erythroid maturation. In primary wild-type (WT) erythroid cells, knockdown of Rad23b or chemical inhibition of the proteasome reduced survival and differentiation capability. Finally, the defects linked to Rad23b loss specifically affected fetal definitive erythropoiesis and stress erythropoiesis in adult mice. Together, these data indicate a previously unappreciated requirement for Rad23b and the UPS in regulation of proliferation in different cell types.