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BACKGROUND AND OBJECTIVES Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples,potentially due to different temperatures during cell staining. MATERIALS AND METHODS Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients,and from 10 paediatric patients. After thawing,cells were prepared for analysis within 30 min between thawing and acquisition,at either 4°C or at room temperature. RESULTS For cell products cryopreserved in conventional freezing medium (10% final DMSO),viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed,CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT),followed by CD3,CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO),median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS The post-thaw environment and,potentially,the cryoprotectant impact the outcome of cell enumeration,and results from the analysis tube may not be representative of the cells infused into a patient. View Publication

Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation. View Publication

BACKGROUND Haemolytic infection lyses red blood cells,releasing haemoglobin (Hb) into the plasma. Although recent studies showed that immune cells recognize redox-active cytotoxic extracellular Hb (metHb) bound to pathogen-associated molecular patterns (PAMPs),currently available information is limited to experiments performed in defined conditions using single cell lines. Therefore,a systemic approach targeting primary whole blood cells is required to better understand the cellular immune defence against metHb and PAMPs,when under a haemolytic infection. METHODS We investigated how human white blood cells,including neutrophils,respond to metHb and lipoteichoic acid (LTA) by measuring reactive oxygen species (ROS),signalling mediators (ERK and p38),NF-κB,cytokines,elastase secretion and cell activation markers. FINDINGS metHb activates NF-κB in TLR2-expressing HEK293 cells but not in normal or TLR9-expressing HEK293 cells. Treatment of isolated neutrophils with metHb increased production of ROS and expressions of IL-8,TNFα,and CD11b,which were further enhanced by metHb + LTA complex. While LTA stimulated the survival of neutrophils,it caused apoptotic cell death when complexed with metHb. The activation of neutrophils by metHb + LTA was subdued by the presence of other types of white blood cells. INTERPRETATION metHb and metHb + LTA complex are ligands of TLR2,inducing an unconventional TLR signalling pathway. Neutrophils are a highly sensitive cell type to metHb + LTA complex. During a haemolytic infection,white blood cells in the vicinity crosstalk to modulate neutrophil TLR-signalling induced by metHb and LTA. View Publication

Biologic TNFα inhibitors are a mainstay treatment option for patients with rheumatoid arthritis (RA) refractory to other treatment options. However,many patients either do not respond or relapse after initially responding to these agents. This study was carried out to identify biomarkers that can distinguish responder from non-responder patients before the initiation of treatment. The level of cytokines in plasma and those produced by ex vivo T cells,B cells and monocytes in 97 RA patients treated with biologic TNFα inhibitors was measured before treatment and after 1 and 3 months of treatment by multiplex analyses. The frequency of T cell subsets and intracellular cytokines were determined by flow cytometry. The results reveal that pre-treatment,T cells from patients who went on to respond to treatment with biologic anti-TNFα agents produced significantly more GM-CSF than non-responder patients. Furthermore,immune cells from responder patients produced higher levels of IL-1β,TNFα and IL-6. Cytokine profiling in the blood of patients confirmed the association between high levels of GM-CSF and responsiveness to biologic anti-TNFα agents. Thus,high blood levels of GM-CSF pre-treatment had a positive predictive value of 87.5% (61.6 to 98.5% at 95% CI) in treated RA patients. The study also shows that cells from most anti-TNFα responder patients in the current cohort produced higher levels of GM-CSF and TNFα pre-treatment than non-responder patients. Findings from the current study and our previous observations that non-responsiveness to anti-TNFα is associated with high IL-17 levels suggest that the disease in responder and non-responder RA patients is likely to be driven/sustained by different inflammatory pathways. The use of biomarker signatures of distinct pro-inflammatory pathways could lead to evidence-based prescription of the most appropriate biological therapies for different RA patients. View Publication

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. View Publication

To investigate retinal involvement in chronic Chagas' disease,we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency,under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors,all related to the same subfamily of G-protein-coupled receptors. View Publication