A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.

A number of malarial blood-stage candidate vaccines are currently being tested in human clinical trials, but our understanding of the relationship between clinical immunity and data obtained from in vitro assays remains inadequate. An in vitro assay which could reliably predict protective immunity in vivo would facilitate vaccine development. Merozoite surface protein1 (MSP1) is a leading blood-stage malaria vaccine candidate, and anti-MSP1 antibodies from individuals that are clinically immune to malaria inhibit the invasion of Plasmodium merozoites into erythrocytes in vitro. Using expression in Escherichia coli and subsequent refolding, we have produced two allelic forms of MSP1 42 (FVO and 3D7). Aotus nancymai monkeys were immunized with MSP1 42 -FVO, MSP1 42 -3D7, or a combination of FVO and 3D7 allelic forms, (MSP1 42 -C1) and were subsequently challenged with Plasmodium falciparum FVO parasites. Sera obtained prior to challenge were tested by standardized enzyme-linked immunosorbent assay (ELISA) to determine antibody titer, and immunoglobulin G (IgG) fractions were also obtained from the same sera; the IgG fractions were tested in an in vitro growth inhibition (GI) assay to evaluate biological activity of the antibodies. Regardless of the immunogen used, all monkeys that had >200,000 ELISA units against MSP1 42 -FVO antigen before challenge controlled their infections. By contrast, all monkeys whose purified IgGs gave <60% inhibition activity in an in vitro GI assay with P. falciparum FVO required treatment for high parasitemia after challenge. There is a strong correlation between ELISA units (Spearman rank correlation of greater than 0.75) or GI activity (Spearman rank correlation of greater than 0.70) and protective immunity judged by various parameters (e.g., cumulative parasitemia or day of patency). These data indicate that, in this monkey model, the ELISA and GI assay values can significantly predict protective immunity induced by a blood-stage vaccine, and they support the use of these assays as part of evaluation of human clinical trials of MSP1-based vaccines.

Plasmodium falciparum invasion of human erythrocytes involves several parasite and erythrocyte receptors that enable parasite invasion by multiple redundant pathways. A key challenge to the development of effective vaccines that block parasite infection of erythrocytes is identifying the players in these pathways and determining their function. Invasion by the parasite clone, Dd2, requires sialic acid on the erythrocyte surface; Dd2/NM is a variant selected for its ability to invade neuraminidase-treated erythrocytes that lack sialic acid. The P. falciparum protein, reticulocyte homology 4 (PfRH4), is uniquely up-regulated in Dd2/NM compared with Dd2, suggesting that it may be a parasite receptor involved in invasion. The aim of the present study was to determine the role of PfRH4 in invasion of erythrocytes and to determine whether it is a target of antibody-mediated blockade and thus a vaccine candidate. We show that both native PfRH4 and a recombinant 30-kDa protein to a conserved region of PfRH4 (rRH430) bind strongly to neuraminidase-treated erythrocytes. rRH430 blocks both the erythrocyte binding of the native PfRH4 and invasion of neuraminidase-treated erythrocytes by Dd2/NM. Taken together, these results indicate that PfRH4 is a parasite receptor involved in sialic acid-independent invasion of erythrocytes. Although antibodies to rRH430 block binding of the native protein to erythrocytes, these antibodies failed to block invasion. These findings suggest that, although PfRH4 is required for invasion of neuraminidase-treated erythrocytes by Dd2/NM, it is inaccessible for antibody-mediated inhibition of the invasion process.erythrocyte invasion ͉ red cell invasion ͉ invasion pathways ͉ erythrocyte binding ͉ sialic acids U nlike Plasmodium vivax, which depends completely on the single interaction of the parasite's Duffy binding protein with the Duffy blood group antigen on erythrocytes (1, 2), Plasmodium falciparum exploits multiple parasite receptors to invade erythrocytes. The redundancy in molecular interactions allows P. falciparum to use alternate pathways for invasion of human erythrocytes. The full repertoire of parasite receptors is not yet identified, and the role in alternate invasion pathways of those identified still remains to be fully defined (3-5).Most of the parasite receptors that are known to play a role in erythrocyte binding and invasion of Plasmodium can be classified into two families. First, the Duffy binding-like (DBL) family that includes the P. vivax/Plasmodium knowlesi Duffy binding proteins and the P. falciparum erythrocyte binding-like proteins (EBA-175, BAEBL, JESEBL, EBL-1, and PEBL). Second, the reticulocyte binding-like (RBL) family that includes the Plasmodium yoelii 235-kDa rhoptry proteins, the P. vivax reticulocyte binding proteins (PvRBP-1 and -2), and the P. falciparum reticulocyte homology (PfRH) proteins (PfRH1, PfRH2a, PfRH2b, PfRH3, PfRH4, and PfRH5) (3-5).Here, we focus on one member of the PfRH family of parasite receptors, P. falciparum reticulocyte homology 4, P...

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between host receptors and parasite ligands (1). Plasmodium vivax and the related simian malaria parasite Plasmodium knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes (2, 3). P. knowlesi can also invade rhesus erythrocytes using alternative Duffy-independent receptors (4). P. falciparum commonly uses sialic acid residues of glycophorin A as invasion receptors (5-9). Like P. knowlesi, P. falciparum also invades erythrocytes by multiple pathways and is not completely dependent on sialic acid residues of glycophorin A (8,10,12,13).Parasite ligands that bind host receptors to mediate erythrocyte invasion include P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi ␤ and ␥ proteins, which bind Duffy-independent receptors on rhesus erythrocytes, and P. falciparum sialic acid-binding protein (also known as EBA-175), which binds sialic acid residues on glycophorin A (4, 14 -18). These parasite ligands share similar features and belong to a family of erythrocyte-binding proteins (19). The extracellular domain of each erythrocyte-binding protein contains two conserved cysteine-rich regions, regions II and VI, at the amino and carboxyl ends, respectively. P. falciparum EBA-175 contains a tandem duplication (F1 and F2) of the N-terminal, conserved, cysteine-rich region. The functional receptor binding domain of each erythrocyte-binding protein lies in region II (20, 21). In the case of EBA-175, region F2 was found to have receptor b...

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The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzymelinked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Objective:Families' perceptions, beliefs, and attitudes about malaria causation, symptom identification, treatment of malaria, and prevention are often overlooked in malaria control efforts. This study was conducted to understand these issues, which can be an important step towards developing strategies, aimed at controlling malaria.Materials and Methods:A community based descriptive cross-sectional study in four villages: Danwarai, Gehuru, Jiga, and Kashin Zama of Aliero local government area in Kebbi Sate, in northern Nigeria. Two hundred household were randomly selected and interviewed using standardized questionnaire.Results:Knowledge of the role of mosquitoes in malaria transmission (11.8%) and cause of malaria (9.6%) was observed to be low among the study population. Comprehensive knowledge about malaria prevention measures was high (90%), but not reflecting in their practice (16%). They have good knowledge of mosquito behavior (breeding areas (64.5%), resting places (70%) and biting time (81%)). Seeking hospital care for a febrile child was a good practice (68.5%) observed. Attitudes regarding the best antimalarial therapy was limited (56.7%) to chloroquine.Conclusions:Misconceptions about malaria transmission and its cause still exist. Knowledge about preventive measures does not necessarily translate into improvement in practices. There is a need for targeted educational programs to increase the communities' efforts to develop desirable attitude and practices regarding malaria and their participation for malaria control.

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