Carla Cerami

The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. We show here that recombinant Plasmodium falciparum circumsporozoite protein (CS) binds specifically to regions of the plasma membrane of hepatocytes exposed to circulating blood in the Disse space. No binding has been detected in other organs, or even in other regions of the hepatocyte membrane. The interaction of CS with hepatocytes, as well as sporozoite invasion of HepG2 cells, is inhibited by synthetic peptides representing the evolutionarily conserved region II of CS. We conclude that region II is a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Our findings provide a rational explanation for the target cell specificity of malaria sporozoites.

Effects of epoetin alfa on the central nervous system

Erythropoietin (EPO) is a glycoprotein that has been shown to mediate response to hypoxia, and is most notably recognized for its central role in erythropoiesis. In a series of experiments using rodent models, the ability of systemically administered recombinant human erythropoietin (r-HuEPO, epoetin alfa) to cross the blood-brain barrier and affect the outcome of neuronal injury or cognitive function was evaluated. It was shown that EPO and EPO receptors are expressed at capillaries of the brain-periphery interface, and that systemically administered epoetin alfa crossed the blood-brain barrier. Compared with control animals, epoetin alfa significantly reduced tissue damage in an ischemic stroke model when administered 24 hours before inducing stroke, with significant protection still evident when epoetin alfa was administered 6 hours poststroke. Epoetin alfa reduced injury by blunt trauma when administered 24 hours before trauma, with a significantly smaller volume of tissue necrosis noted when compared with controls. The observation that epoetin alfa may reduce nervous system inflammation was confirmed when an experimental autoimmune encephalomyelitis model in which rats were shown to have significantly delayed onset and reduced severity of experimental autoimmune encephalomyelitis symptoms after treatment with epoetin alfa. Epoetin alfa also was shown to ameliorate the latency and severity of seizures, and significantly increase survival versus controls when exposed to kainate. These findings suggest future potential therapeutic uses for epoetin alfa beyond its current use to increase erythropoiesis.

Binding of malarial circumsporozoite protein to sulfatides [Gal(3-SO4)β1-Cer] and cholesterol-3-sulfate and its dependence on disulfide bond formation between cysteines in region II

Region II of the malaria circumsporozoite (CS) protein is highly conserved between the CS proteins of different species of malaria. Amino acid sequences homologous to that of region II are found in thrombospondin, properdin, von Willebrand factor and a few other proteins. We show here that the native CS protein from the rodent parasite Plasmodium berghei, and recombinant Plasmodium vivax and Plasmodium falciparum CS proteins containing region II, but not recombinant proteins lacking region II, specifically bind to sulfatides and cholesterol-3-sulfate. The binding is abolished following reduction and alkylation of the proteins. Region II contains 2 cysteines separated by only 3 amino acids, S(N), V, T, and these are the only cysteines present in our recombinant proteins. Therefore, our findings strongly suggest that the region II cysteines are linked by a disulfide bond forming a small peptide loop. We also present evidence that the recognition of sulfatides, cholesterol-3-sulfate, or other cross-reactive sulfated macromolecules by region II may be required during sporozoite invasion of liver cells. Antibodies to a peptide representing region II react with live sporozoites and with sporozoites fixed with glutaraldehyde, indicating that this region is exposed on the surface of the parasites. Furthermore, we have found that the sulfatide and cholesterol-3-sulfate recognition by the CS proteins, and the invasion of hepatocytes by P. berghei sporozoites, are specifically inhibited by dextran sulfate.

High-performance liquid chromatographic method for guanylhydrazone compounds

A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti-inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 micrograms/ml using a 100 microliters injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats.

Epoetin alfa has potential efficacy in central nervous system disorders

Recombinant human erythropoietin (rHuEPO, epoetin alfa) is a 165-amino acid glycoprotein that has been shown to initiate response to hypoxia and is most widely known for its efficacy in the treatment of a variety of anemias. A series of experiments were conducted using rodent models to investigate the ability of systemically administered epoetin alfa to cross the blood-brain barrier (BBB) and affect the outcome of hypoxia and injury in the central nervous system. Results demonstrated that endogenous erythropoietin (EPO) and EPO receptors are expressed around animal brain capillaries and that systemically administered epoetin alfa crossed the BBB. The epoetin alfa group experienced significantly reduced (P <0.01) tissue damage in an ischemic stroke model when the study drug was administered 24 h before inducing stroke compared with control animals. Significant protective effects of epoetin alfa persisted when epoetin alfa was administered up to 6 h poststroke (P <0.05). Similarly, epoetin alfa reduced trauma-related brain injury when administered 24 h prior to and up to 6 h after blunt trauma when compared with the control group. The volume of tissue necrosis was significantly greater in control animals compared with those that received epoetin alfa (P < 0.05). In addition, these studies led to the postulate that epoetin alfa may also have an effect on nervous system inflammation. This was confirmed using an experimental auto-immune encephalomyelitis model, where rats were shown to have significantly delayed onset (P <0.01) and reduced severity (P < 0.05) of symptoms after treatment with epoetin alfa. Other studies demonstrated that epoetin alfa had an effect on the latency and severity of seizures and significantly increased (P <0.0002) survival versus controls when mice were exposed to the neurotoxin kainate (used to induce seizures). These findings suggest future potential therapeutic uses for epoetin alfa beyond its anemia-related effects.