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Biomedical subjects

L H Miller

Publications and source records attributed to L H Miller.

At least 109 records · Page 6Linked to original sources
PubMed1990

The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites.

Plasmodium vivax and Plasmodium knowlesi merozoites invade human erythrocytes that express Duffy blood group surface determinants. A soluble parasite protein of 135 kd binds specifically to a human Duffy antigen. Using antisera affinity purified on the 135 kd protein, we cloned a gene that encodes a member of a P. knowlesi family of erythrocyte binding proteins. The gene is a member of a family that includes three homologous genes located on separate chromosomes. Two genes are expressed as major membrane-bound products that give rise to soluble erythrocyte binding proteins: the 135 kd Duffy binding protein and a 138 kd protein that binds only rhesus erythrocytes. These different erythrocyte binding specificities may result from sequence divergence of the homologous genes. The Duffy receptor family is localized in micronemes, an organelle found in all organisms of the phylum Apicomplexa.

Amino Acid Sequence↗
PubMed1990

Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes.

Irradiated malaria sporozoites can induce CD8+ T cells that are required for protection against infection. However, the parasite antigens targeted by this immune response are unknown. We have discovered a 16-amino acid epitope from the Plasmodium yoelii circumsporozoite (CS) protein that is recognized by cytotoxic T cells from immune mice. Lymphocytes stimulated with this peptide can kill P. yoelii liver stage parasites in vitro in an MHC-restricted, antigen-specific manner. Thus, epitopes from the CS protein are presented on the surface of infected hepatocytes and can be targets for T cells, even though intact CS protein has not been detected on the surface of the infected hepatocyte. A vaccine that induced CTL to parasite antigens might protect humans against malaria by eliminating liver stage parasites.

Animals↗
PubMed1990

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria.

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.

Animals↗
PubMed1990

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites.

Immunoelectron microscopy demonstrated the presence of Pf155/RESA in dense granules of Plasmodium falciparum merozoites rather than in micronemes as previously suggested. Since the dense granules are released after the merozoite enters the parasitophorous vacuole, the role of Pf155/RESA in invasion and subsequent steps of parasite development may differ from that of a molecule located in the micronemes.

Animals↗
PubMed1990

Cellular mechanisms in immunity to blood stage infection.

We studied mechanisms of immunity to blood stage infection in the mouse malarias Plasmodium vinckei and Plasmodium yoelii 17X. Infection with P. vinckei was uniformly lethal, whereas P. yoelii 17X caused a self-limited, nonlethal infection. Transfer of immune CD4+ T cells conferred protection against P. yoelii in nude mice. Previous studies by others had suggested that immunity to P. yoelii may be related to MHC class I expression on reticulocytes and found that CD8+ T cells alone transferred protection in immunodeficient mice. However, in our experiments, immune CD8+ T cells failed to transfer protection. In the P. vinckei system, both B cell-deficient and immunologically intact mice developed immunity to P. vinckei after parasite infection and drug cure. In vivo depletion of CD4+ T cells abrogated immunity in these immune mice. Adoptive transfer of CD4+ T cells failed to protect nude or normal mice from P. vinckei infection, but the transfer of immune CD4+ T cells reconstituted immunity in CD4-depleted immune mice. Splenectomy of immune mice resulted in the complete loss of immunity. Despite the fact that immunity to P. vinckei could be achieved with live parasite infection and drug cure, immunization of mice with killed P. vinckei with various adjuvants failed to protect mice from live challenge. In contrast, immunization with killed P. vinckei antigens in combination with attenuated Salmonella typhimurium SL3235 induced a high degree of protective immunity. These results suggest that induction of immunity against virulent malarias requires both induction of CD4+ T cells and certain splenic alterations caused by parasite infection or S. typhimurium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region.

We investigated Plasmodium falciparum parasitized erythrocyte binding to proteolytic fragments of thrombospondin and the effects of anti-thrombospondin monoclonal antibodies on this binding. Purified human platelet thrombospondin was cleaved by trypsin, chymotrypsin or thrombin. Fragments were separated by heparin-agarose affinity chromatography, removing the amino-terminal heparin-binding region. Trypsin at 5.0 micrograms ml-1 of thrombospondin cleaved thrombospondin to reduced 140 and 120 kDa fragments plus a reduced 25-kDa heparin-binding fragment. Infected erythrocytes bound to intact thrombospondin (3420 +/- 460 infected erythrocytes mm-2) and the carboxy-terminal fragment, yielding 120-140-kDa fragments on sulfhydryl reduction, but not to the 25-kDa fragment (144 +/- 104 infected erythrocytes mm-2 (mean +/- s.d., N = 4). Similar results were obtained with chymotrypsin and thrombin cleavage. When the anti-thrombospondin monoclonal antibody MA-I was added to immobilized thrombospondin prior to infected erythrocytes, adherence was inhibited by 99%. At the same concentration, MA-I inhibited adherence to C32 melanoma cells by only 35%. MA-I binds to a calcium-dependent structure at the C-terminal globular region of thrombospondin. Monoclonal antibody MA-II inhibited adherence to thrombospondin by 46%, while MA-III had no effect. These antibodies bind to the N-terminal globular region which includes the heparin-binding site and the segment connecting the two globular regions, respectively. The site(s) for infected erythrocyte binding on thrombospondin reside in the large, 140- or 120-kDa, proteolytic cleavage fragments, and not in the N-terminal heparin-binding region.

Animals↗
PubMed1990

Method for in situ hybridization to polytene chromosomes from ovarian nurse cells of Anopheles gambiae (Diptera: Culicidae).

A procedure for in situ hybridization to polytene chromosomes from the ovarian nurse cells of Anopheles gambiae Giles has been developed. This procedure involves a modification of established methods for Drosophila larval salivary gland polytene chromosomes. Treatment of chromosome squashes with xylene followed by slow rehydration provides required resolution of chromosome banding patterns, possibly because fatty contaminants are removed from ovarian nurse cell preparations. Using this procedure, unique DNA sequences from a genomic library of An. gambiae have been mapped on adult mosquito polytene chromosomes. The ability to locate genetic markers on chromosomes will allow correlation of physical and genetic maps. Such maps will facilitate identification of genetic loci and expand our knowledge of the genomic organization of An. gambiae.

Animals↗
PubMed1990

Immunization of mice against Plasmodium vinckei with a combination of attenuated Salmonella typhimurium and malarial antigen.

Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.

Animals↗
PubMed1990

Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.

Animals↗
PubMed1990

Evidence implicating MHC genes in the immunological nonresponsiveness to the Plasmodium falciparum CS protein.

The circumsporozoite (CS) protein is a major candidate vaccine antigen for the sporozoite stage of malaria. Both cytotoxic T cells (CTL) and antibody specific for the CS protein are thought to be important in protection. By examining the immune response in mice and humans we have shown that genes mapping to the major histocompatibility complex (MHC) are important for immune responsiveness. F1 mice between high antibody responders and low antibody responders are high antibody responders, suggesting that in this model immune suppressor genes do not control the immune response. Using synthetic peptides to map epitopes for CTL and helper T cells (which are important for the antibody response) we have shown that the T-cell epitopes are located in the polymorphic region of the protein, and we hypothesize that T cells have indeed selected the variation observed in the CS protein. The success of subunit vaccines will depend on the pattern of variation in different geographical locations, the ability to construct multivalent vaccines containing different variant epitopes from this protein, and on the existence of other sporozoite and liver-stage proteins involved in protection.

Animals↗
PubMed1990

A CTL epitope on the circumsporozoite protein of P. yoelii.

Humans are infected with malaria by the bite of anophelene mosquitos carrying plasmodia sporozoites. These sporozoites pass quickly from the blood into hepatocytes, where they develop into mature liver-stage parasites over several days. The clinical stage of the illness begins only when the liver-stage parasites rupture into the bloodstream and erythrocytes are invaded. The pre-erythrocytic stages of malaria are inviting targets for vaccine development, because an effective immune response to these early stages would prevent symptomatic infections.

Amino Acid Sequence↗
PubMed1990

Genetic restriction of protective immunity to Plasmodium yoelii sporozoites.

Ten congenic strains of mice were immunized with irradiated sporozoites of Plasmodium yoelii. When challenged with viable sporozoites, only two strains had a high proportion of animals which did not develop blood-stage infections. Genes both within and outside the H-2 region affected the degree of protection. Immunity did not correlate with anti-sporozoite antibody levels. In vivo depletion of CD8+ T cells did not alter immunity in two of three congenic strains, implying the existence of a novel mechanism of cell-mediated immunity.

Animals↗
PubMed1989

Genetic control of immunity to Plasmodium yoelii sporozoites.

Using a rodent malaria system, we have shown that protective immunity to the preerythrocytic stages of malaria is genetically controlled by MHC and non-MHC genes. Ten congenic strains of mice were immunized with irradiated sporozoites of Plasmodium yoelii. When challenged with viable sporozoites, only two strains had a high proportion of animals that did not develop blood stage infections. Immunity did not correlate with antisporozoite antibody levels. Two protective mechanisms exist determined by non-H-2 genes, and each mechanism is further controlled by H-2-linked Ir genes. On the BALB background only H-2d mice are protected, and protection is abolished by depleting CD8+ T cells. In contrast, on the B10 background only H-2q mice are strongly protected, and protection is not affected by CD8+ T cell depletion. If similar complex genetic regulation of immunity occurs in the human malarias, it will be a major hurdle for vaccine development.

Animals↗
PubMed1989

Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development.

We studied immunity to the blood stage of the rodent malaria, Plasmodium vinckei vinckei, which is uniformly lethal to mice. BALB/c mice develop solid immunity after two infections and drug cure. The following experiments define the basis of this immunity. Transfer of pooled serum from such immune mice renders very limited protection to BALB/c mice and no protection to athymic nu/nu mice. Moreover, B cell-deficient C3H/HeN mice develop immunity to P. vinckei reinfection in the same manner as immunologically intact mice, an observation made earlier. In vivo depletion of CD4+ T cells in immune mice abrogates their immunity. This loss of immunity could be reversed through reconstitution of in vivo CD4-depleted mice with fractionated B-, CD8-, CD4+ immune spleen cells; however, adoptive transfer of fractionated CD4+ T cells from immune spleen into naive BALB/c or histocompatible BALB/c nude mice does not render recipients immune. In vivo depletion of CD8+ T cells did not influence the parasitemia in nonimmune or immune mice. Splenectomy of immune mice completely reverses their immunity. Repletion of splenectomized mice with their own spleen cells does not reconstitute their immunity. We conclude that some feature of the malaria-modified spleen acts in concert with the effector/inducer function of CD4+ T cells to provide protection from P. vinckei. To be consistent with this finding, a malaria vaccine may require a combination of malaria Ag to induce immune CD4+ T cells and an adjuvant or other vaccine vehicle to alter the spleen.

Animals↗