Merozoite surface protein 1, immune evasion, and vaccines against asexual blood stage malaria

There is an urgent need for a vaccine against malaria and proteins on the surface of the merozoite are good targets for development as vaccine candidates because they are exposed to antibody. However, it is possible that the parasite has evolved mechanisms to evade a protective immune response to these proteins. Merozoite surface protein 1 (MSP-1) is a candidate for vaccine development and its C-terminal sequence is the target of protective antibody. MSP-1 is cleaved by proteases in two processing steps, the second step releases the bulk of the protein from the surface and goes to completion during successful red blood cell invasion. Antibodies binding to the C-terminus of Plasmodium falciparum MSP-1 can inhibit both the processing and erythrocyte invasion. Other antibodies that bind to either the C-terminal sequence or elsewhere in the molecule are 'blocking' antibodies, which on binding prevent the binding of the inhibitory antibodies. Blocking antibodies are a mechanism of immune evasion, which may be based on antigenic conservation rather than diversity. This mechanism has a number of implications for the study of protective immunity and the development of malaria vaccines, emphasising the need for appropriate functional assays and careful design of the antigen.     

T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice

Diseases caused by gammaherpesviruses such as Epstein-Barr virus are a major health concern, and there is significant interest in developing vaccines against this class of viral infections. However, the requirements for effective control of gammaherpesvirus infection are only poorly understood. The recent development of the murine herpesvirus MHV-68 model provides an experimental tool to dissect the immune response to gammaherpesvirus infections. In this study, we investigated the impact of priming T cells specific for class I- and class II-restricted epitopes on the acute phase of the infection and the subsequent establishment of latency and infectious mononucleosis. The data show that vaccination with either major histocompatibility complex class I- or class II-restricted T-cell epitopes derived from lytic cycle proteins significantly reduced lung viral titers during the acute infection. Moreover, the peak level of latently infected spleen cells was significantly reduced following vaccination with immunodominant CD8(+) T-cell epitopes. However, this vaccination approach did not prevent the long-term establishment of latency or the development of the infectious mononucleosis-like syndrome in infected mice. Thus, the virus is able to establish latency efficiently despite strong immunological control of the lytic infection.     

Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1

Erythrocyte invasion by the malaria merozoite is prevented by serine protease inhibitors. Various aspects of the biology of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1), including the timing of its expression and its apical location in the merozoite, suggest that this enzyme is involved in invasion. Recombinant PfSUB-1 expressed in a baculovirus system is secreted in the p54 form, noncovalently bound to its cognate propeptide, p31. To understand the role of p31 in PfSUB-1 maturation, we examined interactions between p31 and both recombinant and native enzymes. CD analyses revealed that recombinant p31 (rp31) possesses significant secondary structure on its own, comparable with that of folded propeptides of some bacterial subtilisins. Kinetic studies demonstrated that rp31 is a fast binding, high affinity inhibitor of PfSUB-1. Inhibition of two bacterial subtilisins by rp31 was much less effective, with inhibition constants 49-60-fold higher than that for PfSUB-1. Single (at the P4 or P1 position) or double (at P4 and P1 positions) point mutations of residues within the C-terminal region of rp31 had little effect on its inhibitory activity, and truncation of 11 residues from the rp31 C terminus substantially reduced, but did not abolish, inhibition. None of these modifications prevented binding to the PfSUB-1 catalytic domain or rendered the propeptide susceptible to proteolytic digestion by PfSUB-1. These studies provide new insights into the function of the propeptide in PfSUB-1 activation and shed light on the structural requirements for interaction with the catalytic domain.     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Clarification and application of an ion parametric resonance model for magnetic field interactions with biological systems

Theoretical models proposed to date have been unable to clearly predict biological results from exposure to low-intensity electric and magnetic fields (EMF). Recently a predictive ionic resonance model was proposed by Lednev, based on an earlier atomic spectroscopy theory described by Podgoretskii and Podgoretskii and Khrustalev. The ion parametric resonance (IPR) model developed in this paper corrects mathematical errors in the earlier Lednev model and extends that model to give explicit predictions of biological responses to parallel AC and DC magnetic fields caused by field-induced changes in combinations of ions within the biological system. Distinct response forms predicted by the IPR model depend explicitly on the experimentally controlled variables: magnetic flux densities of the AC and DC magnetic fields (B_ac and B_dc, respectively); AC frequency (f_ac); and, implicitly, charge to mass ratio of target ions. After clarifying the IPR model and extending it to combinations of different resonant ions, this paper proposes a basic set of experiments to test the IPR model directly which do not rely on the choice of a particular specimen or endpoint. While the fundamental bases of the model are supported by a variety of other studies, the IPR model is necessarily heuristic when applied to biological systems, because it is based on the premise that the magnitude and form of magnetic field interactions with unhydrated resonant ions in critical biological structures alter ion-associated biological activities that may in turn be correlated with observable effects in living systems. © 1994 Wiley-Liss, Inc.     

Nonbinomial distributions of relative neurite outgrowth in PC-12 cells

Previously we reported the results of a series of experimental tests using PC-12 cells to examine the biological effects of prescribed combinations of both nerve growth factor and magnetic fields. Because our assay of the PC-12 cells is based on a binary classification of the cells following treatment, our data might be expected to have a binomial distribution. However, our data consistently show a smaller variability than that predicted by the binomial distribution model. In this paper, we examine some possible reasons for this reduction in variability in our results. © 1996 Wiley-Liss, Inc.     

Double blind test of magnetic field effects on neurite outgrowth

Previous work reported that nerve growth factor-stimulated neurite outgrowth in PC-12 cells could be altered by exposure to parallel alternating current (AC) and direct current (DC) magnetic fields under a variety of exposure conditions, producing results that are consistent with the predictions of the ion parametric resonance (IPR) model. The credibility of these results, considered extraordinary by some scientists, could be strengthened if the cell response were found to persist under alternate assay conditions. We replaced part of our standard assay procedure with a double blind procedure. This new procedure obscured 1) whether a particular set of dishes of cells was exposed or not, and 2) which individual dish was in which exposure system. The goal was to determine whether the previously observed responses of PC-12 cells to magnetic fields would be sufficiently robust to decode the imposed blinding, thereby removing any question of experimenter bias in reported results. We placed three coded dishes of cells in each of two otherwise identical exposure systems, one not energized and one energized to produce exposure conditions predicted to maximally suppress neurite outgrowth (B_dc of 36.6 μT, parallel 45 Hz AC of 23.8 μT rms). Each of the six dishes were recoded before assay to further obscure the exposure identity of any individual dish. The combined results of four distinct runs of these double blind experiments unequivocally demonstrated that 1) there was a clear, distinctive, repeatable consistency with the actual energization of the exposure systems and location of each dish, and with the predictions of the IPR model; 2) only the explicitly stated experimental variables influenced the experiment; and 3) the reported response of the cells was very improbably due to chance (P = .000024). Bioelectromagnetics 19:204–209, 1998. © 1998 Wiley-Liss, Inc.

1 This article was prepared by a group consisting of both United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.     

Selective modulation of endothelial cell [Ca2+]i response to flow by the onset rate of shear stress

The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration ([Ca2+]i) increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime.