Recovery of adhesion to chondroitin-4-sulphate in Plasmodium falciparum varCSA disruption mutants by antigenically similar PfEMP1 variants.

Protection against maternal malaria has been associated with the acquisition of a specific antibody response that prevents adhesion of Plasmodium falciparum-infected erythrocytes to the glycosaminoglycan chondroitin-4-sulphate (CSA), which is present in the placental intervillous space. These antibodies are directed against variant forms of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that mediate binding to CSA. We have generated insertional disruption mutants of the gene encoding the CSA-binding phenotype in the P. falciparum clone FCR3 (varCSA) to test the hypothesis that strategies targeting the parasite's determinant for this adhesive phenotype may prevent sequestration of infected erythrocytes in the placenta and hence the development of maternal malaria. The varCSA-disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA. We show that recovery of CSA binding is varCSA independent and mediated by the activation of a novel var variant. Importantly, the corresponding PfEMP1 protein reacts with a monoclonal antibody recognizing the DBL3 gamma domain of the varCSA gene product, indicating that the DBL3 gamma CSA-binding domains are conserved between these PfEMP1-binding variants. Our data support strategies exploring these conserved epitopes as vaccine candidates against maternal malaria.

Illumination of the malaria parasite Plasmodium falciparum alters intracellular pH. Implications for live cell imaging.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasite Plasmodium falciparum, including pH homeostasis, Ca(2+) signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

Acetylcholine increases the free intracellular calcium concentration in podocytes in intact rat glomeruli via muscarinic M(5) receptors.

The effects of acetylcholine (ACh) on the free intracellular calcium concentration ([Ca2+](i)) of microdissected glomeruli were investigated using fura-2 fluorescence digital imaging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca2+](i) increases with an initial peak followed by a sustained plateau, which was suppressed by reduced extracellular Ca2+ concentrations. The [Ca2+](i) plateau was not affected by the L-type Ca2+ channel blocker nicardipine, whereas gadolinium and lanthanum (both at 1 microM) blocked the plateau. Diphenylacetoxy-N:-methylpiperidine methiodide (100 nM), an M(3)/M(5) receptor antagonist, and pirenzepine (1 microM), an M(1) receptor antagonist, completely inhibited the effect of ACh. [Ca2+](i) measurements using two-photon excitation of fluo-3 and staining of the cells with calcein/acetoxymethyl ester, for observation of the capillary network together with the glomerular cells, showed that [Ca2+](i) was increased in single podocytes. Immunohistochemical studies did not demonstrate M(3) receptor expression in glomerular cells. M(1) receptors could be detected only in the parietal sheet of Bowman's capsule, whereas M(5) receptors were found only in podocytes. The data show that ACh increases [Ca2+](i) in podocytes of intact glomeruli, most likely via muscarinic M(5) receptors.