© F e r r a t a S t o r t i F o u n d a t i o nphoid T FH , cT FH cells can express significant levels of surface programmed cell death-1 (PD-1), 31 although the function of PD-1 on T FH cells remains controversial since it is associated with both promotion 16,17 and inhibition of B-cell responses. 18,[32][33][34] Taken together, these reports underscore the need for better characterization of markers for cT FH cells displaying defined functions not only in steady state but also in diseases. Remarkably, PD-1 has been described as a member of the growing family of inhibitory receptors also referred to as immune checkpoints, responsible for aborting T-cell responses. 35 Interestingly, another member of the immune checkpoint family, TIGIT (T-cell immunoreceptor with Ig and immunoreceptor tyrosinebased inhibitory domains), was reported to be overexpressed on both tonsillar and cT FH cells, 17 and was shown to be involved in interactions between T cells and follicular dendritic cells to regulate B-cell responses. 36,37 However, the functional activity of TIGIT on T FH cells, including cT FH cells, has not been studied to date.In this study, we took the approach of using TIGIT and PD-1 to characterize the phenotype and function of circulating T FH subsets and to investigate whether expression of these molecules on cT FH cells modulated their functions in healthy volunteer donors and in a group of chronically transfused SCD patients with or without alloantibodies. Methods Human samplesAll studies were approved by the Institutional Review Boards of the New York Blood Center (NYBC). De-identified fresh leukopaks were obtained from healthy donors at the NYBC. For SCD patients' samples, blood was obtained solely from discard apheresis waste bags obtained during erythrocytapheresis procedures at the Children's Hospital of Philadelphia (see Online Supplementary Material for details). T-cell studiesFreshly-sorted CD4 + T-cell subsets and autologous naïve or memory B cells were used (see Online Supplementary Material for details). Blocking antibodies for TIGIT 38 and PD-1 34,39 were preincubated with sorted T cells before being co-cultured with autologous B cells. Results PD- Expression of ICOS, CD40L and IL-21 by TIGIT + cT FH cellsWe next tested whether TIGIT + cT FH cells were functionally different from cT FH + cT FH populations from a small number of healthy donors (n=3 or 4) were sorted and their ability to express T FH -associated co-stimulatory markers and cytokines following stimulation was compared to those of sorted autologous PD-1 -/TIGIT -subsets (gating strategy shown in Online Supplementary Figure S1). As a control, sort-purified autologous CXCR5 -non-cT FH cells expressing TIGIT E. Godefroy et al. 1416haematologica | 2015; 100(11) A B C© F e r r a t a S t o r t i F o u n d a t i o n ("TIGIT+") or not ("TIGIT-") were also tested. We first monitored expression of co-stimulatory molecules CD40L and ICOS, both specialized in providing Bcell help, on T-cell subsets before or after stimulation by immunos...

Babesia parasites cause a malaria-like febrile illness by infection of red blood cells (RBCs). Despite the growing importance of this tick-borne infection, its basic biology has been neglected. Using novel synchronization tools, the sequence of intra-erythrocytic events was followed from invasion through development and differentiation to egress. The dynamics of the parasite population were studied in culture, revealing for the first time, the complete array of morphological forms in a precursor-product relationship. Important chronological constants including Babesia's highly unusual variable intra-erythrocytic life cycle, the life span of each population of infected cells and the time required for the genesis of the different parasite stages were elucidated. Importantly, the maintenance of specific ratios of the infected RBC populations was shown to be responsible for the parasites' choice of developmental pathways, enabling swift responses to changing environmental conditions like availability of RBCs and nutrition. These results could impact the control of parasite proliferation and therefore disease.

In tropical areas, where unsupervised use of antimalarial drugs is common, patients with an illness consistent clinically with severe malaria but with negative blood smears pose a management dilemma. Malaria pigment is evident in peripheral blood leukocytes in greater than 90% of patients with severe malaria. To characterize the clearance kinetics of parasitized erythrocytes and malaria pigment-containing leukocytes, sequential peripheral blood and intradermal smears were assessed in 27 adult Vietnamese patients with severe falciparum malaria. The clearance of parasitized erythrocytes and pigment-containing monocytes (PCMs) followed first order kinetics. The elimination of pigment-containing neutrophils (PCNs) was first order initially, but deviated from this when counts were low. Clearance of peripheral blood PCMs (median clearance time, 216 hours; range, 84 to 492 hours) was significantly slower than that of parasitized erythrocytes (median, 96 hours; range, 36 to 168 hours) or PCNs (median, 72 hours; range, 0 to 168 hours; P < .0001). Intradermal PCM clearance times were the longest of all (median, 12 days; range, 6 to 23 days; significantly longer than peripheral blood PCM clearance, P < .001). Twenty-one (88%) patients still had signs, symptoms, or laboratory features of severe malaria after parasite clearance but before phagocyte pigment clearance. Sixteen of the 23 surviving patients (70%; 95% confidence interval, 50% to 87%) still had intraleukocytic malaria pigment on peripheral blood films 72 hours after parasite clearance. Thus, by determining the distribution of malaria pigment in peripheral blood and intradermal phagocytes, the time since effective antimalarial treatment started can be estimated. Microscopy for intraleukocytic pigment is valuable in the differential diagnosis of severe febrile illnesses in malarious areas where uncontrolled use of antimalarial drugs is widespread.

Apoptosis, which leads to phagocytosis by mononuclear cells, represents the primary mechanism for removing neutrophils from inflamed tissues and minimizing injury. The present studies show that membrane phosphatidylserine turnover and permeability, as well as DNA fragmentation, were reduced in neutrophils from neonates when compared with adults. The activity of caspase 3 and expression of the proapoptotic proteins Bax, Bad, and Bak were also decreased in neonatal relative to adult neutrophils. These findings are consistent with impaired apoptosis in neonatal cells, which may contribute to prolonged inflammation in infants after oxidative stress or infection. Neutrophil apoptosis is induced by endogenous ligands such as Fas (FasL), which engage death receptors of the tumor necrosis factor/nerve growth factor superfamily, including Fas receptor (FasR). We found that expression of FasR was decreased in neonatal when compared with adult cells. Moreover, neonatal neutrophils did not undergo apoptosis in response to anti-FasR antibody and exhibited impaired chemotaxis to soluble FasL. However, in both adult and neonatal cells, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase inhibitors blocked Fas-induced activity. These data suggest that prolonged survival of neonatal neutrophils at injured sites is due, in part, to reduced responsiveness to FasL. This may be related to decreased expression of both FasR and Bcl-2-family proteins that mediate neutrophil apoptosis. Human neutrophils are the primary effector cells in acute inflammation and are rapidly recruited from the bloodstream to injured sites. Although neutrophils from newborns exhibit defects in chemotaxis, phagocytosis, and oxidative metabolism (1-4), infants are at high risk for neutrophil-mediated tissue injury. Activated neutrophils are cleared from inflamed sites by the process of apoptosis, followed by macrophage phagocytosis. This promotes resolution rather than persistence of tissue injury (5,6). Attenuation of neutrophil apoptosis in neonates may contribute to severe and prolonged inflammatory responses. In premature infants, this may play a role in conditions such as bronchopulmonary dysplasia and necrotizing enterocolitis, which result in significant mortality and morbidity.Neutrophil longevity and functional activity are regulated by inflammatory mediators present in the microenvironment.Whereas proinflammatory cytokines, such as interferon-␥ (IFN-␥), granulocyte-monocyte colony-stimulating factor (GM-CSF), and bacterial-derived lipopolysaccharide (LPS), promote neutrophil activation and survival, anti-inflammatory mediators, such as IL-10 (IL-10) and Fas ligand (FasL), are proapoptotic (7-10). These mediators regulate the expression of pro-and antiapoptotic mitochondrial proteins, including Bak, Bad, Bax, and A1, as well as the proapoptotic effector protease caspase 3 (10 -14). Previous studies have demonstrated that the rate of apoptosis is reduced in peripheral blood neutrophils from neonates relative to adult cells (15,16). W...