Summary Artemisinin and its derivatives (ARTs) are frontline antimalarial drugs. However, ART monotherapy is associated with a high frequency of recrudescent infection, resulting in treatment failure. A subset of parasites is thought to undergo ART-induced latency, but the mechanisms remain unknown. Here we report that ART treatment results in phosphorylation of the parasite eukaryotic initiation factor-2α (eIF2α), leading to repression of general translation and latency induction. Enhanced phosphorylated eIF2α correlates with high rates of recrudescence following ART, and inhibiting eIF2α dephosphorylation renders parasites less sensitive to ART treatment. ART-induced eIF2α phosphorylation is mediated by the Plasmodium eIF2α kinase, PK4. Overexpression of a PK4 dominant-negative or pharmacological inhibition of PK4 blocks parasites from entering latency and abolishes recrudescence after ART treatment of infected mice. These results show that translational control underlies ART-induced latency and that interference with this stress response may resolve the clinical problem of recrudescent infection.

There is significant evidence that brain-infiltrating CD8+ T cells play a central role in the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the mechanisms through which they mediate their pathogenic activity during malaria infection remain poorly understood. Utilizing intravital two-photon microscopy combined with detailed ex vivo flow cytometric analysis, we show that brain-infiltrating T cells accumulate within the perivascular spaces of brains of mice infected with both ECM-inducing (P. berghei ANKA) and non-inducing (P. berghei NK65) infections. However, perivascular T cells displayed an arrested behavior specifically during P. berghei ANKA infection, despite the brain-accumulating CD8+ T cells exhibiting comparable activation phenotypes during both infections. We observed T cells forming long-term cognate interactions with CX3CR1-bearing antigen presenting cells within the brains during P. berghei ANKA infection, but abrogation of this interaction by targeted depletion of the APC cells failed to prevent ECM development. Pathogenic CD8+ T cells were found to colocalize with rare apoptotic cells expressing CD31, a marker of endothelial cells, within the brain during ECM. However, cellular apoptosis was a rare event and did not result in loss of cerebral vasculature or correspond with the extensive disruption to its integrity observed during ECM. In summary, our data show that the arrest of T cells in the perivascular compartments of the brain is a unique signature of ECM-inducing malaria infection and implies an important role for this event in the development of the ECM-syndrome.

Summary Plasmodium species, the parasitic agents of malaria, invade erythrocytes to reproduce resulting in erythrocyte loss. However, a greater loss is caused by the elimination of uninfected erythrocytes, sometimes long after infection has been cleared. Using a mouse model, we found that Plasmodium infection induces the generation of anti-self antibodies that bind to the surface of uninfected erythrocytes from infected, but not uninfected, mice. These antibodies recognize phosphatidylserine, which is exposed on the surface of a fraction of uninfected erythrocytes during malaria. We find that phosphatidylserine-exposing erythrocytes are reticulocytes expressing high levels of CD47, a ‘do-not-eat-me’ signal, but the binding of anti-phosphatidylserine antibodies mediates their phagocytosis, contributing to anemia. In human patients with late post-malarial anemia, we found a strong inverse correlation between the levels of anti-phosphatidylserine antibodies and plasma hemoglobin, suggesting a similar role in humans. Inhibition of this pathway may be exploited for treating malarial anemia.

Atherosclerosis is a chronic disease that affects medium and large arteries. This process originates from the interaction between cells of the arterial wall, lipoproteins and inflammatory cells, leading to the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in acute clinical complications such as myocardial infarction and stroke. Owing to the heterogeneous cellular composition of the plaques, a proteomic analysis of the whole lesion is not appropriate. Therefore, we have studied the proteins secreted by human carotid atherosclerotic plaques, obtained by endarterectomy. Normal artery segments and different regions of the surgical pieces (noncomplicated plaque, complicated plaque with thrombus) were cultured in protein-free medium and the secreted proteins (supernatants) analyzed by two-dimensional gel electrophoresis. Normal artery segments secreted a moderate number of proteins (42 spots). However in the two-dimensional (2-D) gels (pH 3-10) of segments bearing a plaque, the number of spots increased markedly (154). The number of spots also increased (202) in the 2-D gels of artery segments with a ruptured plaque and thrombus. Thus, the more complicated the lesion, the higher the number of secreted proteins, suggesting the production of specific proteins relating to the complexity of the atherosclerotic lesion.

Abstract-IgG Fc receptors (Fc␥Rs) play a role in activating the immune system and in maintaining peripheral tolerance, but their role in atherosclerosis is unknown. We generated double-knockout (DKO) mice by crossing apolipoprotein E-deficient mice (apoE Ϫ/Ϫ ) with Fc␥R ␥ chain-deficient mice (␥ Ϫ/Ϫ ). The size of atherosclerotic lesions along the aorta was approximately 50% lower in DKO compared with apoE Ϫ/Ϫ control mice, without differences in serum lipid levels. The macrophage and T-cell content of lesions in the DKO were reduced by 49Ϯ6% and 56Ϯ8%, respectively, compared with the content in apoE Ϫ/Ϫ lesions. Furthermore, the expression of monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated on Activated Normal T-cell Expressed and Secreted), and intercellular adhesion molecule-1 (ICAM-1) and the activation of nuclear factor-B (NF-B) were significantly reduced in aortic lesions from DKO mice. In vitro, vascular smooth muscle cells (VSMCs) from both ␥ Ϫ/Ϫ and DKO mice failed to respond to immune complexes, as shown by impaired chemokine expression and NF-B activation. ApoE Ϫ/Ϫ mice have higher levels of activating Fc␥RI and Fc␥RIIIA, and inhibitory Fc␥RIIB, compared with wild-type mice. The DKO mice express only the inhibitory Fc␥RIIB receptor. We conclude that Fc␥R deficiency limits development and progression of atherosclerosis. In addition to leukocytes, Fc␥R activation in VSMCs contributes to the inflammatory process, in part, by regulating chemokine expression and leukocyte invasion of the vessel wall. These results underscore the critical role of Fc␥Rs in atherogenesis and support the use of immunotherapy in the treatment of this disease. Key Words: Fc receptors Ⅲ atherosclerosis Ⅲ double-knockout mice Ⅲ immune complexes A therosclerosis is a chronic inflammatory disease of the arterial wall characterized by progressive accumulation of lipids, cells, and extracellular matrix. In recent years, the immune processes associated to atherogenesis have received considerable attention. Studies in both humans and animals have demonstrated that atheromatous lesions at all stage of development contain a wide variety of cells and molecules characteristic of immune system, such as macrophages, lymphocytes, CD40, interferon-␥, major histocompatibility complex-II, complement, and antibodies (Abs). [1][2][3][4] In addition, congenital deficiency of macrophages, T and B lymphocytes, or even the inhibition of their mediators has resulted in the reduction of atherosclerotic lesion. 1,3 One of the critical steps in atherogenesis is the accumulation within the arterial intima of cholesteryl ester-laden foam cells, many of them derived from macrophages, whose formation is ultimately dependent on the uptake of various forms of low-density lipoproteins (LDLs). 1,2 Although emphasis has been placed on scavenger receptors, foam-cell development may also be influenced by lipoprotein interaction with other receptors, such as LDL receptors, very-low-density lipoprotein (VLDL) receptors, and IgG Fc (Fc␥Rs) receptors. 5 Diff...

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum-infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter-endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin-induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC-induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised.

Apoptosis is implicated in the progressive cell loss and fibrosis both at glomerular and tubulointerstitial level. In this study, we examined the potential mechanisms by which persistent proteinuria (protein-overload model) could induce apoptosis. After uninephrectomy (UNX), Wistar rats received daily injections of 0.5 g of bovine serum albumin (BSA)/100 g body weight or saline. Both at day 8 and day 28, rats receiving BSA had proteinuria and renal lesions characterized by tubular atrophy and/or dilation and mononuclear cell infiltration. In relation to control-UNX rats, renal cortex of nephritic rats showed an increment in AT2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) expression. In both groups, AT2 receptor immunostaining was mainly localized in proximal tubular cells. Rats with persistent proteinuria showed a significantly increased number of terminal dUTP nick-end labeling positive apoptotic cells compared with UNX-controls, both in glomeruli and tubulointerstitium. Double staining for apoptosis and AT2 receptor showed that most terminal dUTP nick-end labeling positive cells were found in tubules expressing AT2 receptor. Using an antibody that recognizes the active form caspase-3, we observed an increment in caspase-3 activation in rats receiving BSA with respect to those receiving saline. Rats with persistent proteinuria showed a diminution in the phosphorylation of Bcl-2 with respect to UNX-controls both at day 8 and day 28. By contrast, no changes were observed either in the Bax or in the Bcl-2 protein levels. The administration of BSA to UNX rats induced a diminution in the phosphorylation of ERK with respect to UNX-control at all times studied. The changes observed in ERK activities took place without alterations of ERK1/2 protein levels. In summary, our data suggest that persistent proteinuria causes apoptosis in tubular cells through the activation of AT2 receptor, which can, in turn, inhibit MAP kinase (ERK1/2) activation and Bcl-2 phosphorylation.

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (γ−/−) were performed. Severe glomerular damage was induced in WT and Wγ chimeras (BM from WT to irradiated γ−/−), while it was absent in γ−/− and γW chimeras (γ−/− BM to WT). Moreover, WT PMN transfer, but not γ−/− PMN, reconstituted the disease in γ−/−, indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-κB activation and TNF-α overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H2O2 inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-κB and TNF-α much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-α expression predominantly via FcR-mediated H2O2 production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.