Srabasti J. Chakravorty

Exported Proteins Required for Virulence and Rigidity of Plasmodium falciparum-Infected Human Erythrocytes

Summary A major part of virulence for Plasmodium falciparum malaria infection, the most lethal parasitic disease of humans, results from increased rigidity and adhesiveness of infected host red cells. These changes are caused by parasite proteins exported to the erythrocyte using novel trafficking machinery assembled in the host cell. To understand these unique modifications, we used a large-scale gene knockout strategy combined with functional screens to identify proteins exported into parasite-infected erythrocytes and involved in remodeling these cells. Eight genes were identified encoding proteins required for export of the parasite adhesin PfEMP1 and assembly of knobs that function as physical platforms to anchor the adhesin. Additionally, we show that multiple proteins play a role in generating increased rigidity of infected erythrocytes. Collectively these proteins function as a pathogen secretion system, similar to bacteria and may provide targets for antivirulence based therapies to a disease responsible for millions of deaths annually.

Fractalkine expression in human renal inflammation

Immune and inflammatory human renal disease is associated with heavy mononuclear cell infiltration. The trafficking of these cells to extravascular sites is directed by local production of chemokines. Fractalkine is the first described cell‐surface anchored chemokine and has potent mononuclear cell‐directed adhesion and chemotactic properties. The purpose of this study was to analyse the expression and distribution of fractalkine in human renal inflammation. In situ hybridization and immunohistochemistry were used to study renal biopsies from 15 patients with predominant glomerular inflammation (vasculitic glomerulonephritis) and 15 with predominant tubular and interstitial inflammation (acute renal allograft rejection). Controls comprised non‐inflammatory glomerulonephritis and normal tissue. Fractalkine mRNA was predominantly expressed in the major compartment, glomerular or tubulointerstitial, affected by disease and with the strongest expression localized to vascular sites local to inflammation. In acute renal allograft rejection, there was increased expression of fractalkine mRNA by tubular epithelial cells. There was no expression of fractalkine by infiltrating leukocytes and there was only sparse expression in control tissue. Fractalkine mRNA expression correlated with infiltrating leukocyte subsets. Immunohistochemistry confirmed this pattern of expression, with serial section co‐localization showing fractalkine expression in areas with macrophage (CD68+) and T cell (CD3+) infiltrates. These expression patterns show that fractalkine is a strong candidate for directing mononuclear cell infiltration in human renal inflammation. Copyright

Von Willebrand factor propeptide in malaria : evidence of acute endothelial cell activation

The pathogenicity of Plasmodium falciparum is thought to relate to the unique ability of infected erythrocytes to adhere to and subsequently activate the vascular endothelium. To study the state of endothelial activation during falciparum malaria, we measured plasma levels of both von Willebrand factor (VWF) and its propeptide, indices of chronic and acute endothelial cell perturbation, respectively. Results were correlated with clinical and biochemical markers of disease severity, including plasma lactate. Our data show that acute endothelial cell activation is a hallmark of malaria in children, indicated by a significant rise in VWF and VWF propeptide. The highest VWF and propeptide levels were seen in cerebral and non‐cerebral severe malaria, and associations found between VWF propeptide level and lactate (P < 0·001). Mean VWF propeptide levels (nmol/l) were in cerebral malaria 33·4, non‐cerebral severe malaria 26·3, mild malaria 22·1, non‐malaria febrile illness 10·2, and controls 10·1. Differences between patient and control groups were highly significant (P < 0·005). Follow‐up of 26 cerebral malaria cases showed that levels of VWF propeptide, but not VWF fell by 24 h, following the clinical course of disease and recovery. These novel findings potentially implicate acute, regulated exocytosis of endothelial cell Weibel–Palade bodies in the pathogenesis of Plasmodium falciparum malaria.

PfEMP1 expression is reduced on the surface of knobless Plasmodium falciparum infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this species of human malarial parasite. PfEMP1 is expressed on the surface of infected erythrocytes (IEs) and directly mediates adhesion to a variety of host cells. A number of other parasite-encoded proteins are similarly exported to the IE plasma membrane and play an indirect role in this adhesion process through the modification of the erythrocyte cytoskeleton and the formation of electron dense knobs into which PfEMP1 is anchored. Analysis of the specific contribution of knob-associated proteins to adhesion is difficult due to rapid PfEMP1 switching during in vitro culture. Furthermore, these studies typically assume that the level and distribution of PfEMP1 exposed in knobby (K+) and knobless (K–) IEs is unaltered, an assumption not yet supported with data. We describe here the preparation and characterisation of a panel of isogenic K+ and K– parasite clones that express one of two defined PfEMP1 variants. Analysis of the cytoadhesive properties of these clones shows that both static and flow adhesion is reduced in all the K– clones and, further, that this correlates with an approximately 50% reduction in PfEMP1 displayed on the IE surface. However, despite this reduction, the gross distribution of PfEMP1 in K– IEs appears unaltered. These data impact on our current interpretation of the role of knobs in adhesion and the mechanism of trafficking PfEMP1 to the IE surface.

Fractalkine expression on human renal tubular epithelial cells: potential role in mononuclear cell adhesion

Fractalkine (CX3CL1) is a transmembrane molecule with a CX3C chemokine domain attached to an extracellular mucin stalk which can induce both adhesion and migration of leucocytes. Mononuclear cell infiltration at renal tubular sites and associated tubular epithelial cell damage are key events during acute renal inflammation following renal allograft transplantation. Using northern and Western blot analysis, we have demonstrated the expression of fractalkine message and protein by renal tubular epithelial cells in vitro. The expression was up‐regulated by TNF‐α, a key proinflammatory cytokine in acute rejection. Investigation of surface expression of fractalkine on cultured proximal tubular epithelial cells revealed only a subpopulation of positively staining cells. Immunohistochemistry revealed that only a proportion of tubules in renal allograft biopsies showed induction of fractalkine expression. Studies using a static model of adhesion demonstrated CX3CR1/fractalkine interactions accounted for 26% of monocytic THP‐1 cell and 17% of peripheral blood natural killer cell adhesion to tubular epithelial cells, suggesting that fractalkine may have a functional role in leucocyte adhesion and retention, at selected tubular sites in acute renal inflammation. Thus, fractalkine blockade strategies could reduce mononuclear cell mediated tubular damage and improve graft survival following kidney transplantation.

Host response to cytoadherence in Plasmodium falciparum

Cytoadherence of PRBCs (Plasmodium falciparum-infected red blood cells) to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying the disease are poorly understood. Endothelial cells have the ability to mobilize immune and pro-adhesive responses when exposed to both PRBCs and TNF (tumour necrosis factor). In addition, there is also an up-regulation by PRBCs and TNF and a concurrent down-regulation of a range of genes involved in inflammation and cell death, by PRBCs and TNF. We propose that the balance between positive and negative regulation will contribute to endothelial pathology during malarial infection. Apposition of PRBCs has been shown by a number of groups to activate signalling pathways. This is dependent, at least in part, on the cytoadherence characteristics of the invading isolate, such that the avidity of the PRBC for the receptor on host endothelium is proportional to the level of activation of the signalling pathways. An understanding of the post-adhesive processes produced by cytoadherence may help us to understand the variable pathology seen in malaria and to design appropriate therapies to alleviate severe disease.

Plasmodium Falciparum Intercellular Adhesion Molecule-1-Based Cytoadherence-Related Signaling in Human Endothelial Cells

BACKGROUND Cytoadherence of Plasmodium falciparum-infected erythrocytes to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying disease are poorly understood. METHODS We compared parasite lines varying in their binding properties to human endothelial cells for their ability to stimulate signaling activity. RESULTS In human umbilical vein endothelial cells (HUVECs), which rely on adhesion to intercellular adhesion molecule (ICAM)-1 for binding, signaling is related to the avidity of the parasite line for ICAM-1 and can be blocked either through the use of anti-ICAM-1 monoclonal antibodies or HUVECs with altered ICAM-1 binding properties (i.e., ICAM-1(Kilifi)). Human dermal microvascular endothelial cells (HDMECs), which can bind infected erythrocytes via ICAM-1 and CD36, have a more complex pattern of signaling behavior, but this is also dependent on adhesive interactions rather than merely contact between cells. CONCLUSIONS Signaling via apposition of P. falciparum-infected erythrocytes with host endothelium is dependent, at least in part, on the cytoadherence characteristics of the invading isolate. An understanding of the postadhesive processes produced by cytoadherence may help us to understand the variable pathologies seen in malaria disease.

Potential role for monocyte chemotactic protein-4 (MCP-4) in monocyte/macrophage recruitment in acute renal inflammation.

The CC chemokine, monocyte chemoattractant protein‐4 (MCP‐4), is an important chemoattractant for monocytes and T cells. Recent data indicate a role in renal inflammation. This study has used in situ hybridization and immunohistochemical analysis of cryostat sections of biopsy material taken from patients with acute renal allograft rejection and vasculitic glomerulonephritis to demonstrate renal expression of MCP‐4, both at message and protein level. MCP‐4 was primarily expressed at peritubular, periglomerular, and perivascular sites, irrespective of the inflammatory condition, and was associated with infiltrating CD3‐positive lymphocytes and CD68‐positive monocyte/macrophages. In addition, proximal tubular epithelial cells grown in culture from cortical fragments of human kidney showed low levels of constitutive MCP‐4 expression, detectable by western blotting; this expression of MCP‐4 was up‐regulated in response to the pro‐inflammatory cytokines, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). CCR3‐, CCR5‐ and CCR2‐expressing leukocyte populations were identified at sites of MCP‐4 expression. Double‐staining techniques revealed that CC chemokine receptor‐expressing cells were primarily CD68‐positive. These studies suggest an important role for MCP‐4 in the recruitment and retention of monocytes/macrophages in renal inflammation. Copyright

Rational Design of Anticytoadherence Inhibitors for Plasmodium falciparum Based on the Crystal Structure of Human Intercellular Adhesion Molecule 1

ABSTRACT Adhesion of Plasmodium falciparum-infected erythrocytes (IE) to host endothelium has been associated with pathology in malaria. Although the interaction with endothelial cells can be complex due to the relatively large number of host receptors available for binding, specific proteins have been identified that are more commonly used than others. For example, binding to intercellular adhesion molecule 1 (ICAM 1) is found frequently in parasites from pediatric cases of malaria. The binding site for P. falciparum-infected erythrocytes on ICAM 1 has been mapped in some detail and is distinct from the site for lymphocyte function-associated antigen 1 (LFA-1). Part of the ICAM 1 binding site for P. falciparum-infected erythrocytes (the DE loop) was used to screen a library of compounds based on its structure (derived from the crystal structure of human ICAM 1). This resulted in the identification of 36 structural mimeotopes as potential competitive inhibitors of binding. One of these compounds, (+)-epigalloyl-catechin-gallate [(+)-EGCG], was found to inhibit IE adhesion to ICAM 1 in a dose-dependent manner with two variant ICAM 1-binding parasite lines, providing the first example of a potential mimeotope-based anticytoadherence inhibitor for Plasmodium falciparum.

T lymphocyte adhesion mechanisms within inflamed human kidney: studies with a Stamper-Woodruff assay.

Renal inflammatory conditions are characterized by mononuclear cell recruitment to sites of inflammation. We have developed a modified Stamper-Woodruff assay system to analyze mechanisms of functional T cell adhesion to cryostat sections of renal biopsy material from patients with vasculitic glomerulonephritis (GN) and acute allograft rejection. Peripheral blood T cells adhered to intraglomerular, periglomerular, and tubulointerstitial regions of the cortex. Blocking monoclonal antibodies against tissue expressed ICAM-1, VCAM-1, and the CS-1 domain of fibronectin (CS-1Fn) differentially attenuated T cell adhesion. Glomerular adhesion in vasculitic GN and tubulointerstitial adhesion in acute rejection were particularly sensitive to both anti-ICAM-1 and anti-VCAM-1 antibodies, indicating a prominent role for ICAM-1 and VCAM-1 at glomerular sites in vasculitis and at tubulointerstitial sites in rejection. Furthermore, using KL/4 cells (LFA-1 expressing) and Jurkat cells (VLA-4 expressing), we demonstrated specific LFA-1/ICAM-1- and VLA-4/VCAM-1-mediated interactions within glomerular and tubulointerstitial compartments. Jurkat cells also adhered to VCAM-1-free sites, and binding was inhibitable by anti-CS-1Fn antibody, thereby demonstrating a role for VLA-4/fibronectin interactions especially at intraglomerular sites in acute rejection where VCAM-1 is notably absent. We therefore propose a prominent functional role for ICAM-1, VCAM-1, and CS-1 domain fibronectin in T cell recruitment to the inflamed kidney.