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Biomedical subjects

L H Miller

Publications and source records attributed to L H Miller.

At least 55 records · Page 3Linked to original sources
PubMed1997

Efficacy of clean v sterile surgical prep kits.

The goal of preoperative skin preparation is to reduce the risk of postoperative wound infections. This study was designed to determine if a difference exists in the residual microbial flora on the skin of surgical patients who are prepped with clean versus sterile prep kits. The researchers randomly assigned 60 ambulatory surgery patients to two preoperative skin preparation groups (i.e., clean prep kits, sterile prep kits) and obtained cultures of the patients' surgical sites at three different times (i.e., before performing standard povidone-iodine scrub-and-paint skin preps, 10 minutes after the completion of the skin preps, immediately after skin closure). They used repeated mixed factorial analyses of variance to compute the differences in residual microbial counts at the patients' surgical sites. There was no difference in the residual microbial skin flora in the patients prepped with clean or sterile skin prep kits. The study results have significant cost-saving implications for health care facilities and surgical patients.

Adult↗
PubMed1997

Comparison of protection induced by immunization with recombinant proteins from different regions of merozoite surface protein 1 of Plasmodium yoelii.

Vaccination with native full-length merozoite surface protein 1 (MSP1) or with recombinant C-terminal peptides protects mice against lethal challenge with virulent malaria parasites. To determine whether other regions of MSP1 can also induce protection, Plasmodium yoelii MSP1 was divided into four separate regions. Each was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The N-terminal fragment began after the cleavage site for the signal sequence and ended in the region comparable to the cleavage site for the C terminus of the 82-kDa peptide of Plasmodium falciparum. This expressed protein was 30 kDa smaller than the predicted peptide. One peptide from the middle region was produced, and the C terminus consisted of a 42-kDa fragment corresponding to the analogous peptide of P. falciparum and a 19-kDa fragment that extended 37 amino acids in the amino-terminal direction beyond the probable cleavage site. To test protection of mice against lethal P. yoelii challenge, three mouse strains (CAF1, BALB/c, and A/J) were vaccinated with each of the four recombinant proteins of MSP1. Mice vaccinated with the C-terminal 19-kDa protein were highly protected (described previously), as were those vaccinated with the 42-kDa protein that contained the 19-kDa fragment. The N-terminally expressed fragment of P. yoelii was not full length because of proteolytic cleavage in E. coli. The GST-82-kDa partial fragments induced some immunity, but the surviving mice still had high parasitemias. Vaccination with the peptide from the middle region of MSP1 gave minimal to no protection. Therefore, in addition to the C-terminal 19- and 42-kDa proteins, the only other fragment to give protection was the 82-kDa protein. The protection induced by the truncated 82-kDa protein was minimal compared with that of the C-terminal fragments.

Animals↗
PubMed1996

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.

Amino Acid Sequence↗
PubMed1996

The domain on the Duffy blood group antigen for binding Plasmodium vivax and P. knowlesi malarial parasites to erythrocytes.

Plasmodium vivax and the related simian malarial parasite P. knowlesi use the Duffy blood group antigen as a receptor to invade human erythrocytes and region II of the parasite ligands for binding to this erythrocyte receptor. Here, we identify the peptide within the Duffy blood group antigen of human and rhesus erythrocytes to which the P. vivax and P. knowlesi ligands bind. Peptides from the NH2-terminal extracellular region of the Duffy antigen were tested for their ability to block the binding of erythrocytes to transfected Cos cells expressing on their surface region II of the Duffy-binding ligands. The binding site on the human Duffy antigen used by both the P. vivax and P. knowlesi ligands maps to a 35-amino acid region. A 34-amino acid peptide from the equivalent region of the rhesus Duffy antigen blocked the binding of P. vivax to human erythrocytes, although the P. vivax ligand expressed on Cos cells does not bind rhesus erythrocytes. The binding of the rhesus peptide, but not the rhesus erythrocyte, to the P. vivax ligand was explained by interference of carbohydrate with the binding process. Rhesus erythrocytes, treated with N-glycanase, bound specifically to P. vivax region II. Thus, the interaction of P. vivax ligand with human and rhesus erythrocytes appears to be mediated by a peptide-peptide interaction. Glycosylation of the rhesus Duffy antigen appears to block binding of the P. vivax ligand to rhesus erythrocytes.

Amidohydrolases↗
PubMed1996

Genetic regulation of protective immune response in congenic strains of mice vaccinated with a subunit malaria vaccine.

The C-terminal 19-kDa, epidermal growth factor-like region of the merozoite surface protein 1 (MSP1) has been used as a vaccine to induce protective immunity to Plasmodium yoelii in mice and to Plasmodium falciparum in monkeys. To analyze the mechanisms and genetic regulation of this MSP1 vaccine-induced protection, we studied the immunologic correlates of protection in H-2 recombinant and congenic mouse strains on the B10 background. Multiple H-2-linked loci were found to contribute, each with a different mechanism. One locus mapped to the I-A region based on the strong protection in C57BL/10 mice compared with intermediate protection in B10.A(4R) mice and the lack of a difference between B10.AKM and B10.MBR mice. Differences in efficacy of passively transferred antisera from vaccinated C57BL/10 vs B10.A(4R) mice indicated that the protection regulated by the I-A locus was at least in part Ab dependent. Two loci mapped to the right of I-A (FE, H-2S, or H-2D) based on a correlation with the number of H-2k loci to the right of I-A in mice that were I-Ak. One effect was Ab independent and may correspond to a possible negative effect of the I-Ek locus. T cells from protected and nonprotected strains differed in their production of IFN-gamma and TNF-alpha following immunization with MSP1(19), but it was unclear how the differential patterns of cytokine expression related to the level of protection. Thus, MSP1(19) vaccine-induced protection is regulated by H-2-linked loci corresponding to two different immune mechanisms. These findings may indicate the need for more than one Ag in a vaccine to protect an HLA-diverse population.

Animals↗
PubMed1996

Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.

Animals↗
PubMed1996

Permeability of intact and dechorionated eggs of the Anopheles mosquito to water vapor and liquid water: a comparison with Drosophila.

As an initial step in the development of cryopreservation methods for Anopheles gambiae embryos, we determined the permeability of intact and dechorionated A. gambiae embryos to water and ethylene glycol and compared those permeabilities with those of Drosophila melanogaster embryos. Most studies were conducted on embryos allowed to develop for 8 h at 26 degrees C or 15 h at 17 degrees C. Intact A. gambiae embryos are some 50 times more permeable to water vapor than are D. melanogaster embryos; e.g., when air dried, half of 15 h/17 degrees C A. gambiae embryos become severely dehydrated in 3. 5-4.0 min, whereas comparably aged D. melanogaster embryos require approximately 4 h or more. Comparable differences between the two species exist with respect to the loss of liquid water under an osmotic driving force. After 90 min in 0.75 M sucrose in 0.26 osmolal D-20 Drosophila medium, 25-40% of intact A. gambiae embryos underwent extensive shrinkage, whereas D. melanogaster embryos showed no shrinkage. The chorion of both species can be removed by a 2.5-min exposure to 50% household bleach (Clorox). Dechorionation increases the rate of water loss two- to sixfold during air drying in both species and during exposure to hyperosmotic sucrose in A. gambiae. Dechorionated D. melanogaster embryos, however, show no shrinkage in hyperosmotic sucrose even after 6 h. The water permeability of dechorionated A. gambiae embryos thus remains many-fold higher than that of dechorionated D. melanogaster embryos. Although moderately permeable to water, neither intact nor dechorionated A. gambiae embryos are permeable to ethylene glycol, as evidenced by the failure of dehydrated embryos to reexpand in 120-180 min in that solution.

Animals↗
PubMed1996

Permeabilization of eggs of the malaria mosquito Anopheles gambiae.

Although Anopheles gambiae embryos that have developed for 15 h at 17 degrees C are slightly permeable to water, they are impermeable to ethylene glycol, the cryoprotectant used in the cryopreservation of Drosophila melanogaster embryos. Success in cryopreservation requires that they be made permeable to protective solutes. Permeabilization of D. melanogaster was achieved by 1) dechorionation with 50% Clorox (household bleach) followed by 2) a water flush; 3) immersion in isopropyl alcohol to remove most extraembryonic water; 4) 2 min air drying to remove most isopropyl alcohol; 5) 90-s exposure to n-heptane containing 0.3% 1-butanol; and 6) 15-s exposure to pure n-heptane. The permeability of A. gambiae embryos was assessed by determining the times required for the initial dehydration of embryos in 1 M ethylene glycol in 260 mOsm Drosophila culture medium (permeability to water) and the times required for their return to normal volume (permeability to ethylene glycol). Based on these criteria, the above D. melanogaster procedure effectively permeabilizes 15 h/17 degrees C to 19 h/17 degrees C A. gambiae embryos. Nearly all collapsed in <5 min, and most returned to normal volume in approximately 40 min. Although permeable, all were killed by the permeabilization procedure. In analyzing the effect of each step on viability, 50% Clorox caused some lethality, and Clorox followed by isopropyl alcohol was 100% lethal. Decreasing the Clorox concentration to 10% still dechorionated eggs, but with reduced toxicity; substitution of a 10% solution of reagent-grade sodium hypochlorite for Clorox further decreased toxicity. The isopropyl alcohol step was also toxic. Consequently, the removal of residual surface water was achieved by substituting air drying for isopropyl alcohol. The drying took place under direct microscope observation and was continued until the embryos began to shrink. The combination of this modified method for dechorionation and controlled air drying before exposure to heptane resulted in permeabilization of all embryos in most runs and in approximately 30% survival of the permeabilized embryos.

Animals↗
PubMed1995

Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.

Amino Acid Sequence↗
PubMed1995

Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes.

Plasmodium falciparum expresses on the host erythrocyte surface clonally variant antigens and ligands that mediate adherence to endothelial receptors. Both are central to pathogenesis, since they allow chronicity of infection and lead to concentration of infected erythrocytes in cerebral vessels. Here we show that expression of variant antigenic determinants is correlated with expression of individual members of a large, multigene family named var. Each var gene contains copies of a motif that has been previously shown to bind diverse host receptors; expression of a specific var gene correlated with binding to ICAM-1. Thus, our findings are consistent with the involvement of var genes in antigenic variation and binding to endothelium.

Amino Acid Sequence↗
PubMed1995

A mutant of melanoma growth stimulating activity does not activate neutrophils but blocks erythrocyte invasion by malaria.

Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.

Alanine↗
PubMed1995

Plasmodium gallinaceum: a refractory mechanism of ookinete killing in the mosquito, Anopheles gambiae.

We have identified a mechanism for refractoriness to a bird malaria, Plasmodium gallinaceum, in the African vector of human malaria, Anopheles gambiae. Oocysts fail to develop in the refractory mosquitoes as a result of ookinete death which occurs within 27 hr of midgut invasion. Ultrastructural studies showed that parasite death occurs while the ookinete lies free in the midgut epithelial cell cytosol, usually surrounded by an organelle-free zone that consists of finely fibrillar material. The mechanism of parasite killing does not involve a previously described refractory mechanism of parasite encapsulation. We selected genetic lines which are refractory and susceptible to midgut infection. Genetic crossing of the lines suggests that the refractory trait is inherited as a single dominant genetic locus. Other loci probably influence oocyst number in susceptible mosquitoes. Intracellular ookinete killing appears to involve a previously unrecognized host defense mechanism against malaria parasites that involves direct destruction of the invading organism.

Animals↗
PubMed1995

Plasmodium gallinaceum: sporozoite invasion of Aedes aegypti salivary glands is inhibited by anti-gland antibodies and by lectins.

There is evidence which suggests that malaria sporozoites recognize mosquito salivary glands by specific receptor-ligand interactions. We are interested in identifying the putative salivary gland receptor(s) for sporozoite invasion. We used an in vivo bioassay for sporozoite invasion of salivary glands. In this assay, purified sporozoites from mature oocytes of Plasmodium gallinaceum were injected into Aedes aegypti mosquitoes and salivary glands were dissected at different time points after injection. One half of the maximum invasion of salivary glands by sporozoites occurred by 6 hr, and salivary gland sporozoite load did not increase further after 24 hr postinjection. This assay was used to determine the effect of experimental treatments with antibodies and lectins at 24 hr postinjection. We raised a rabbit polyclonal antiserum against female Ae. aegypti salivary glands which recognized tissue-specific determinants in the basal lamina of salivary glands. Purified IgG antibody fraction of the immune serum blocked sporozoite invasion in vivo. We tested a panel of 19 lectins and found 7 which bound to salivary glands. Of these 7, succinylated wheat germ agglutinin and wheat germ agglutinin completely blocked sporozoite invasion; Pisum sativum agglutinin and soybean agglutinin partially blocked; and concanavalin A, Dolichos biflorus agglutinin, and Phaseolus vulgaris erythroagglutinin did not block. Our results suggest that sporozoites interact with glycosylated salivary gland surface molecules which serve as receptors for invasion, and which may be in the salivary gland basal lamina. Because the putative sporozoite receptors contain immunogenic determinants, it is feasible to identify them by an immunological strategy.

Aedes↗
PubMed1995

Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain.

The merozoite cap protein-1 (MCP-1) of Plasmodium falciparum follows the distribution of the moving junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted transmembrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP-1 is divided into three domains. The N-terminal domain includes a 52-amino-acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido-reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP-1 are not found in any other members of this protein family and may reflect the specific function of MCP-1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C-terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C-terminal domain resembles domains in some cytoskeleton-associated proteins and may mediate the interaction of MCP-1 with cytoskeleton in Plasmodium.

Amino Acid Sequence↗