Small angle light scattering assay for the detection of malaria infection.

The diagnosis of malaria, caused by Plasmodium spp., still remains a challenging process. Especially in low-income countries, a rapid user-friendly method is needed for the efficient care of the patient. A small-angle light scattering device consisting of hardware and software was developed. Using the DNA-binding dye SYBR Green, malaria infections could be distinguished in healthy red blood cells infected with Plasmodium. Subsequently, samples from parasite positive and negative patients living in a hyper-endemic area of Kinshasa, DRC were assessed. The scatter profiles were distinct and malaria infection could be detected using the Giemsa stain. Although these results are preliminary, they indicate that the device has the potential to be used as a new diagnostic tool for the detection of Malaria infection.

Direct and indirect bacterial killing functions of neutrophil defensins in lung explants.

Studies of the antimicrobial activity of neutrophil defensins have mostly been carried out in microbiological media, and their effects on the host defense in physiological conditions are unclear. We examined 1) the antibacterial activity of defensins in physiological media with and without lung tissue present, 2) the effect of defensins on hydrogen peroxide (H(2)O(2)) production by lung tissue that had been exposed to bacteria, and 3) the effect of diphenyleneiodonium (DPI), an inhibitor of reactive oxygen species formation, on the antibacterial activity of defensins in the presence of lung tissue. Defensins were incubated with Escherichia coli or Pseudomonas aeruginosa in the absence or presence of primary cultured mouse lung explants. Defensins reduced bacterial counts by approximately 65-fold and approximately 25-fold, respectively, at 48 h; bacterial counts were further decreased by approximately 600-fold and approximately 12,000-fold, respectively, in the presence of lung tissue. Defensins induced H(2)O(2) production by lung tissue, and the rate of killing of E. coli by defensins was reduced by approximately 2,500-fold in the presence of 10 microM DPI. We conclude that defensins exert a significant antimicrobial effect under physiological conditions and that this effect is enhanced in the presence of lung tissue by a mechanism that involves the production of reactive oxygen species.

Characterization of peroxynitrite-oxidized low density lipoprotein binding to human CD36.

Peroxynitrite-mediated oxidation may be an important physiological mechanism for oxidation of low density lipoprotein (LDL), however, the molecular basis for the interaction of peroxynitrite oxidized LDL (OxLDL) with scavenger receptors such as CD36, has not been characterized. In this study, we compared the biochemical characteristics and receptor binding of LDL that was oxidized using: (1) Cu2+, a standard method of oxidizing LDL in vitro; and (2) 3-morpholinosydnonimine (SIN-1), a source of peroxynitrite. Both methods of oxidation caused an increase in electrophoretic migration of LDL, but greater mobility was observed with Cu2+-OxLDL. In addition, greater fragmentation of apolipoprotein B was observed following Cu2+ oxidation than after SIN-1 oxidation. The levels of lipid peroxides and thiobarbituric acid reactive substances were similar after 20 h of oxidation by both methods, although the time-course was distinct. Cu2+ and SIN-1-OxLDL bound specifically to the macrophage scavenger receptor CD36 with high affinity. Binding of the 20 h SIN-1 treated LDL to CD36 was comparable to a 4 h Cu2+ modified LDL. The binding of Cu2+ and SIN-1-OxLDL to CD36 was similar under different biochemical conditions and modifications of the receptor, suggesting that OxLDL particles, generated by either method, bind to the same domain of CD36. The results demonstrate that SIN-1 produced an oxidized LDL particle that binds specifically to CD36 and suggests that peroxynitrite OxLDL may represent a more physiologically relevant model than Cu2+-OxLDL for studying the interactions of OxLDL with cells and lipoprotein receptors in vitro.

Development of monoclonal antibodies which specifically recognize Entamoeba histolytica in preserved stool samples.

We report the generation of monoclonal antibodies against a recombinant 170-kDa subunit of the Gal or GalNAc lectin of Entamoeba histolytica that specifically recognize E. histolytica but not Entamoeba dispar in preserved stool samples. These antibodies do not cross-react with other bowel protozoa, including Entamoeba coli, Giardia lamblia, and Dientamoeba fragilis.

Nonylphenolethoxylates as malarial chloroquine resistance reversal agents.

Malaria-associated morbidity and mortality are increasing because of widespread resistance to one of the safest and least expensive antimalarials, chloroquine. The availability of an inexpensive agent that is capable of reversing chloroquine resistance would have a major impact on malaria treatment worldwide. The interaction of nonylphenolethoxylates (NPEs, commercially available synthetic surfactants) with drug-resistant Plasmodium falciparum was examined to determine if NPEs inhibited the growth of the parasites and if NPEs could sensitize resistant parasites to chloroquine. NPEs inhibited the development of the parasite when present in the low- to mid-micromolar range (5 to 90 microM), indicating that they possess antimalarial activity. Further, the presence of <10 microM concentrations of NPEs caused the 50% inhibitory concentrations for chloroquine-resistant lines to drop to levels (< or =12 nM) observed for sensitive lines and generally considered to be achievable with treatment courses of chloroquine. Long-chain (>30 ethoxylate units) NPEs were found to be most active in P. falciparum, which contrasts with previously observed maximal activity of short-chain ( approximately 9 ethoxylate units) NPEs in multidrug-resistant mammalian cell lines. NPEs may be attractive chloroquine resistance reversal agents since they are inexpensive and may be selectively directed against P. falciparum without inhibiting mammalian tissue P glycoproteins. Antimalarial preparations that include these agents may prolong the effective life span of chloroquine and other antimalarials.

Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein bind to separate domains of CD36.

The cytoadherence of erythrocytes (red blood cells) infected with Plasmodium falciparum (pRBCs) to endothelial cells and the uptake of oxidized low-density lipoprotein (oxLDL) by macrophages are both mediated, in part, by the glycoprotein receptor CD36. The interaction of lipoproteins and pRBCs competing for the human CD36 receptor was examined by use of Chinese hamster ovary cells expressing human CD36. OxLDL competitively inhibits the adherence of pRBCs to CD36, but native LDL and high-density lipoprotein do not. Modification of Lys residues in CD36 inhibits both oxLDL and pRBC binding; however, only oxLDL binding is inhibited by receptor iodination, and only pRBC binding is influenced by pH variations and receptor reduction. Furthermore, peptide inhibitors of the pRBC/CD36 interaction do not influence oxLDL binding. These results suggest that, although oxLDL competitively inhibits the adherence of pRBCs, these ligands interact with distinct domains on the CD36 receptor.

The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36.

CD36 is an 88-kD glycoprotein involved in the cytoadherence of Plasmodium falciparum-parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36.

Band 3 peptides block the adherence of sickle cells to endothelial cells in vitro.

Malaria-parasitized erythrocytes have increased endothelial adherence due to exposure of previously buried intramembranous sites of band 3. Because sickle erythrocytes also show increased adhesiveness and because the membrane portion of band 3 is aggregated in both types of cells, we examined the role of band 3 in sickle cell adhesiveness. Synthetic peptides derived from the second and third exofacial, interhelical regions of band 3 completely inhibited the abnormal adherence of sickle cells to an endothelial monolayer in a static assay. This effect was observed independently of plasma factors, required micromolar levels of peptide, was sequence-specific, and was found with both L- and D-isomers. The active peptides also inhibited the increased adherence induced by low-dose calcium loading of normal red blood cells. Finally, a monoclonal antibody against an active peptide specifically immunostained a fraction of sickle cells. These findings implicate a role for band 3 in at least one type of sickle cell adhesiveness via the exposure of normally cryptic membrane sites.

The effect of proteases and iodination on the adherent behaviour of Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes were treated with proteases (trypsin, chymotrypsin, pronase, or V8 protease) or iodinated and the effect of these treatments on the cytoadherent behaviour of the cells was determined. As previously observed, protease treatment reduced cytoadherence. However, it was also found that the P. falciparum-induced adhesin, pfalhesin, was not removed by protease treatment. Gelatin flotation experiments and scanning electron microscopical examination of the treated cells indicated that protease exposure resulted in changes in the knob structures on the cells, which are known to affect the adherent behaviour of the cells. Iodination was found to be an effective method of inactivating pfalhesin.

The human anion transport protein, band 3, contains a CD36-like binding domain for Plasmodium falciparum-infected erythrocytes.

Epitope mapping of a murine monoclonal antibody (mAb), 5H12, prepared against live Plasmodium falciparum-infected red blood cells indicated that the epitope consisted of amino acid residues 474-487 of the human anion transport protein, band 3. mAb 5H12 enhanced cytoadherence, but inhibited the CD36-like mediated rosetting. A synthetic peptide based on the sequence of the epitope (FSFCETNGLE) blocked both rosetting and cytoadherence, suggesting that this amino acid sequence may form the CD36-like receptor. The CD36-like region of band 3 was antigenically distinct from platelet or endothelial CD36.

Plasmodium falciparum: naturally occurring rabbit immunoglobulins recognize human band 3 peptide motifs and malaria-infected red cells.

The reactivity to overlapping decapeptides based on sequences of human band 3 protein was determined for 24 rabbit serum samples. A high percentage of the sera recognized sequences of amino acid 650-670, as well as several other sequences representing putative exofacial regions of the band 3 protein. When sera were used to stain immunofluorescently human erythrocytes, some of which were infected with P. falciparum, those samples that predominantly reacted with amino acids 650-670 stained P. falciparum-infected red cells, whereas those that reacted with other regions of band 3 stained all erythrocytes. A positive correlation was found between anti-band 3 reactivity and the capacity of a serum to inhibit the cytoadherence of P. falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Also, some rabbit sera immunoprecipitated a high-molecular-weight protein from extracts of surface iodinated P. falciparum-infected red cells.

Plasmodium falciparum: the adherence of erythrocytes infected with human malaria can be mimicked using pfalhesin-coated microspheres.

Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542-555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by immunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells transfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546-553 and 820-829 and called pfalhesin) were produced. The pfalhesin-coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546-553 had little binding capacity for melanoma cells. These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel 'artificial' P. falciparum-infected erythrocytes for use in in vitro and in vivo studies.

The sticky secrets of sequestration.

Sequestration, the adherence of infected erythrocytes containing the more mature stages of parasite development (trophozoites and schizonts) to the endothelial cells lining the capillaries and post-capillary venules, is characteristic of Plasmodium falciparum infections. In this review, Irwin Sherman and his colleagues discuss recent advances in the characterization of the adhesive molecules on the surface of malaria-infected erythrocytes and the receptors on the endothelium to which they bind.

Plasmodium falciparum: sera of individuals living in a malaria-endemic region recognize peptide motifs of the human erythrocyte anion transport protein.

Specific regions of the human erythrocyte anion transport protein, AE 1 or band 3, have been identified as being adhesive in Plasmodium falciparum-infected-erythrocytes. In addition, synthetic peptides based on these sequences and murine monoclonal antibodies (Mabs) to these block cytoadherence/sequestration. These findings suggest the possibility that humans who are able to control P. falciparum infections may produce anti-adhesin (and thus anti-band 3) antibodies. To test this hypothesis, sera from individuals living in The Gambia and southern California were assayed for reactivity to decapeptides patterned on putative exofacial regions of the human anion transporter, band 3 protein. Sera from an area highly endemic for malaria, The Gambia, were found to have strong reactivity to well-defined regions of the band 3 protein (some of which contain the adhesin), whereas minimal reactivity was observed with sera from individuals living in a geographic area where malaria transmission is rare (California). Sera from The Gambia reacted strongly with residue blocks 534-560, 638-660, and 808-842. Gambian sera that reacted strongly with peptides patterned on band 3 failed to react with native band 3 on an immunoblot, but did react with fixed P. falciparum-infected erythrocytes. Using reactivity to decapeptides has allowed the determination of the epitopes of previously described murine MAbs that inhibit or promote cytoadherence; this reactivity with a specific region of a protein can be correlated with a specific effect on cytoadherence. A MAb (5H12) directed against amino acids 474-488 promoted cytoadherence, whereas those directed against amino acids 540-550 and 750-766 (Mabs 1C4 and 4A3, respectively) inhibited cytoadherence.

Monoclonal antibodies that react with human band 3 residues 542-555 recognize different conformations of this protein in uninfected and Plasmodium falciparum infected erythrocytes.

A monoclonal antibody generated against synthetic peptides patterned on amino acids 542-555 of human band 3, designated 1F4, specifically immunostained Plasmodium falciparum-infected erythrocytes and inhibited the cytoadherence of P. falciparum-infected erythrocytes to C32 amelanotic melanoma cells. 1F4 did not recognize intact band 3 protein on immunoblots, however it was reactive towards proteolytic fragments of band 3. The binding region of another murine monoclonal antibody previously reported to recognize the membrane spanning domain of human band 3, designated B6, was found to also recognize residues 542-555, however its properties differed from 1F4. Mab B6 recognized both infected and uninfected red cells, and reacted only with intact band 3 on immunoblots. Mab B6 was without effect on cytoadherence. These results demonstrate that monoclonal antibodies reactive against a common peptide sequence may bind to different conformations of the peptide sequence and suggest that the adherent competency of P. falciparum-infected erythrocytes may result from a change in the surface topography of human band 3 protein.

Plasmodium falciparum: CD36 dependent cytoadherence or rosetting of infected erythrocytes is modulated by knobs.

A knobless (K-) line of the FCR-3 isolate of Plasmodium falciparum was obtained by gelatin flotation. Immunofluorescent staining and immunoblots indicated that both the K- line and the K+ (knobby) line from which it was derived contained similar forms of potentially adhesive modified band 3 protein. When the K+ and K- lines were assayed for their cytoadherent and rosetting abilities the K+ line showed a high level of CD36 dependent cytoadherence, whereas the K- line demonstrated a marked pH dependent increase in rosetting. Rosetting was inhibited by the addition of peptides based on band 3 motifs, suggesting that cytoadherence and rosetting involve the same adhesin but that the presence of knobs affects whether the adherent preference of the infected erythrocyte is uninfected red cells or endothelial/C32 amelanotic melanoma cells.

Immune responses to band 3 neoantigens on Plasmodium falciparum-infected erythrocytes in subjects living in an area of intense malaria transmission are associated with low parasite density and high hematocrit value.

During the intracellular development of the human malarial parasite, Plasmodium falciparum, cryptic regions of the erythrocyte band 3 protein are exposed. Antibodies against these band 3-related neoantigens block cytoadherence, and peptides based on amino acid sequences of putative exofacial loops of band 3 protein block the in vitro and in vivo adherence of P. falciparum-infected erythrocytes. At present, it is not known whether reactivity to these antigens is related to exposure to the malaria parasite or is correlated with protective immunity. The reactivities of plasma to peptides containing amino acid sequences of putative exofacial loops 3 and 7 of human band 3 protein were determined for children and adults living in an area of perennial malaria transmission (Liberia) and for donors who had never been exposed to malaria (Denmark). Plasma samples from children and adults living in an area of intense malaria transmission showed a much higher reactivity with the band 3 peptides than did those from nonimmune individuals. High reactivity to the loop 3 peptide (amino acids 546 to 555) was correlated with lower mean parasite density in children in the 5- to 9-year-old age group. The presence of antibodies against loop 3 and 7 peptides was not associated with a low packed erythrocyte volume (hematocrit); in fact, higher-than-average reactivities to both peptides were positively correlated with high hematocrit values, indicating that antibodies which specifically recognize the band 3-related neoantigens are not involved in hemolysis (autoimmunity).

Antibodies to synthetic peptides based on band 3 motifs react specifically with Plasmodium falciparum (human malaria)-infected erythrocytes and block cytoadherence.

Rabbit polyclonal and mouse monoclonal antibodies (Mabs) prepared against synthetic peptides patterned on exofacial loops 3 (amino acids 546-555) and 7 (821-834) of the human anion transport protein band 3 inhibited the cytoadherence of Plasmodium falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Mabs directed against exofacial loop 4 (amino acids 628-642) did not inhibit adherence to a significant degree. The murine Mabs recognized only P. falciparum-infected cryptic in uninfected erythrocytes.

Cytoadherence-related neoantigens on Plasmodium falciparum (human malaria)-infected human erythrocytes result from the exposure of normally cryptic regions of the band 3 protein.

Murine monoclonal antibodies (Mabs) were produced by vaccination of Balb/c mice with live Plasmodium falciparum-infected red cells (iRBC). The iRBC Mabs recognized altered forms of the human erythrocyte membrane protein band 3; however, these Mabs did not recognize the band 3 molecule in uninfected or ring-infected red cells. The location of epitopes was determined by studying the binding of the iRBC Mabs after selective proteolysis of band 3 as well as by the reactivity of these Mabs to synthetic peptides that corresponded to putative exofacial regions of band 3. Treatment of uninfected red cell membranes with trypsin under low ionic strength conditions resulted in exposure of cryptic epitopes of band 3 which were recognized by the iRBC Mabs. Several of the anti-iRBC Mabs (two of which were described previously) inhibited the in vitro adherence of infected erythrocytes to C32 amelanotic melanoma cells. A mouse polyclonal serum against a synthetic peptide based on an amino acid sequence motif of band 3 reacted (by immunostaining) only with the surface of iRBC and blocked adhesion. Thus, it appears that cryptic residues of the band 3 protein become exposed upon parasitization, and their presence contributes to the increased adhesiveness of the P. falciparum-infected red cell.