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Biomedical subjects

L H Miller

Publications and source records attributed to L H Miller.

At least 73 records · Page 4Linked to original sources
PubMed1995

Cellular mechanisms in the immune response to malaria in Plasmodium vinckei-infected mice.

Infection of mice with the malaria parasite Plasmodium vinckei vinckei is 100% lethal. However, after two infections followed by drug cure, BALB/c mice develop a solid immunity which is antibody independent but mediated by CD4+ T cells. To elucidate the mechanisms of this immunity, spleen cells from immune mice were challenged in vitro with lysates of P. vinckei-infected or uninfected erythrocytes. The parasite antigen induced proliferation of T cells from immune mice but not from nonimmune mice. When gamma interferon production by cells from immune mice was assayed at the single-cell level, 1 to 3 cells per 1,000 cells were found to release this cytokine when exposed to antigen. In contrast, the numbers of interleukin 4 (IL-4)-producing cells from both immune and control mice were < or = 4 per 10(6) cells, regardless of antigen exposure. Investigation in a bioassay showed that P. vinckei antigen induced the release of IL-4 from spleen cells of immune mice but not from those of control mice. Nevertheless, that IL-4 is of minor significance in this system is also suggested by the absence of elevation of immunoglobulin E levels in blood samples from these mice, in contrast to what is seen with P. chabaudi infection, in which IL-4-producing Th2 cells are of major importance for immunity during later phases of infection. Taken together, the present results indicate that immunity to P. vinckei is a Th1 response, with gamma interferon being an important protective factor. Whether or not the Th1 response, through overproduction of tumor necrosis factor alpha, is also responsible for pathology and death in this infection remains to be clarified.

Animals↗
PubMed1995

Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys.

BACKGROUND: The carboxy-terminus of the merozoite surface protein-1 (MSP1) of Plasmodium falciparum has been implicated as a target of protective immunity. MATERIALS AND METHODS: Two recombinant proteins from the carboxy-terminus of MSP1, the 42 kD fused to GST (bMSP1(42)) and the 19 kD (yMSP1(19)), were expressed in Escherichia coli and secreted from Saccharomyces cerevisiae, respectively. To determine if vaccination with these recombinant proteins induces protective immunity, we conducted a randomized, blinded vaccine trial in two species of Aotus monkeys, A. nancymai and A. vociferans. After three injections using Freund's adjuvant, the monkeys were challenged with the virulent Vietnam Oak Knoll (FVO) strain of P. falciparum. RESULTS: All three control monkeys required treatment by Day 19. Two of three monkeys vaccinated with bMSP1(42) required treatment by Day 17, whereas the third monkey controlled parasitemia for 28 days before requiring treatment. In contrast, both of the A. nancymai vaccinated with yMSP1(19) self-resolved an otherwise lethal infection. One of the two yMSP1(19)-vaccinated A. vociferans had a prolonged prepatent period of > 28 days before requiring treatment. No evidence of mutations were evident in the parasites recovered after the prolonged prepatent period. Sera from the two A. nancymai that self-cured had no detectable effect on in vitro invasion. CONCLUSIONS: Vaccination of A. nancymai with yMSP1(19) induced protective immune responses. The course of recrudescing parasitemias in protected monkeys suggested that immunity is not mediated by antibodies that block invasion. Our data indicate that vaccine trials with the highly adapted FVO strain of P. falciparum can be tested in A. nancymai and that MSP1(19) is a promising anti-blood-stage vaccine for human trials.

Amino Acid Sequence↗
PubMed1994

Analysis of human T cell clones specific for conserved peptide sequences within malaria proteins. Paucity of clones responsive to intact parasites.

T cells are thought to be of central importance in malaria immunity. Peptides copying malaria protein sequences often stimulate human CD4+ T cells and it was thought that they represented T cell epitopes present in the parasite and may thus have particular relevance to malaria vaccine development. To verify whether synthetic peptides representing highly conserved regions of parasite Ags may contribute to a malaria vaccine, we searched the data bank for conserved regions of Plasmodium falciparum malaria proteins that were not homologous to known self (human) proteins. We synthesized 24 such peptides representing 11 of the cloned and sequenced malaria asexual stage Ags, which were predicted by algorithms to represent T cell epitopes, and 6 peptides not predicted to be T cell epitopes and used these to generate T cell clones from individuals with an extensive previous history of malaria exposure. The T cell clones responded vigorously to many peptides but only a single clone, specific for a peptide within merozoite surface protein-1, 20-39, VTHESYQELVKKLEALEDAV, and not previously defined to be a T cell epitope responded to malaria parasites by proliferation and secretion of IFN-gamma. This epitope was not revealed by studying parasite-induced T cell lines and is thus subdominant. The clone was able to significantly inhibit parasite growth in vitro. The final step in the inhibition of parasite growth appears to be nonspecific because other activated clones (not specific for malaria sequences) can inhibit parasite growth. Our data suggest that few conserved peptides within malaria parasites can be processed from the intact parasite. However, such peptides that can be processed from malaria parasites may be expected to stimulate parasite-specific T cells that could inhibit parasite growth and as such may be lead candidates for a vaccine aimed at inducing cellular immunity to malaria.

Adult↗
PubMed1994

Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion.

Plasmodium vivax and the related monkey malaria, P. knowlesi, require interaction with the Duffy blood group antigen, a receptor for a family of chemokines that includes interleukin 8, to invade human erythrocytes. One P. vivax and three P. knowlesi proteins that serve as erythrocyte binding ligands in such interactions share sequence homology. Expression of different regions of the P. vivax protein in COS7 cells identified a cysteine-rich domain that bound Duffy blood group-positive but not Duffy blood group-negative human erythrocytes. The homologous domain of the P. knowlesi proteins also bound erythrocytes, but had different specificities. The P. vivax and P. knowlesi binding domains lie in one of two regions of homology with the P. falciparum sialic acid binding protein, another erythrocyte binding ligand, indicating conservation of the domain for erythrocyte binding in evolutionarily distant malaria species. The binding domains of these malaria ligands represent potential vaccine candidates and targets for receptor-blockade therapy.

Animals↗
PubMed1994

Malaria pathogenesis.

Malaria is a disease caused by repeated cycles of growth of the parasite Plasmodium in the erythrocyte. Various cellular and molecular strategies allow the parasite to evade the human immune response for many cycles of parasite multiplication. Under certain circumstances Plasmodium infection causes severe anemia or cerebral malaria; the expression of disease is influenced by both parasite and host factors, as exemplified by the exacerbation of disease during pregnancy. This article provides an overview of malaria pathogenesis, synthesizing the recent field, laboratory, and epidemiological data that will lead to the development of strategies to reduce mortality and morbidity.

Anemia↗
PubMed1994

Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum.

A 175-kilodalton erythrocyte binding protein, EBA-175, of the parasite Plasmodium falciparum mediates the invasion of erythrocytes. The erythrocyte receptor for EBA-175 is dependent on sialic acid. The domain of EBA-175 that binds erythrocytes was identified as region II with the use of truncated portions of EBA-175 expressed on COS cells. Region II, which contains a cysteine-rich motif, and native EBA-175 bind specifically to glycophorin A, but not to glycophorin B, on the erythrocyte membrane. Erythrocyte recognition of EBA-175 requires both sialic acid and the peptide backbone of glycophorin A. The identification of both the receptor and ligand domains may suggest rational designs for receptor blockade and vaccines.

Animals↗
PubMed1994

Impact of malaria on genetic polymorphism and genetic diseases in Africans and African Americans.

The high mortality from malaria in sub-Sahara Africa selected multiple genes that give the population a selective advantage. Identification of the genetic basis for resistance may suggest unusual approaches to development of malarial vaccines and antimalarial drugs. Some of these genes may be deleterious, although of selective advantage within the African setting, and need to be identified for counseling for disease prevention.

Africa South of the Sahara↗
PubMed1994

Plasmodium gallinaceum: differential lysis of two developmental stages of malaria sporozoites by the alternative pathway of complement.

During sporogonic development of Plasmodium gallinaceum in the mosquito vector, two developmentally distinct sporozoite stages can be isolated. Sporozoites obtained from oocysts in abdomens of mosquitoes 10 days after an infective blood meal are poorly infectious to the vertebrate host (chicken); days later, sporozoites isolated from mosquito salivary glands are highly infectious. In a first step toward understanding the physiologic basis of this developmentally regulated infectivity to the vertebrate host, we determined the relative resistance of the two sporozoite stages to lysis by the complement system of the vertebrate host. Whereas 86% of oocyst sporozoites were lysed when incubated in fresh chicken serum in vitro, only 24% of salivary gland sporozoites were lysed under identical incubation conditions. Preincubation of oocyst sporozoites in a homogenate of female Aedes aegypti salivary glands did not diminish their susceptibility to lysis by serum, indicating that lysis was mediated by the interaction of serum factors with parasite-specific molecules. The lytic activity of fresh chicken serum was abrogated by incubating for 45 min at 56 degrees C or chelating with EDTA. Fresh chicken serum specifically depleted of Ca2+, by chelating with EGTA in the presence of Mg2+, retained its ability to lyse oocyst sporozoites. The lysis of salivary gland sporozoites mediated by chicken antisporozoite serum is abrogated by EGTA, indicating that the antibody-dependent complement pathway in chicken serum is blocked by EGTA. Because oocyst sporozoite lysis by serum was heat sensitive and Mg2+ dependent, but Ca2+ independent, we conclude that lysis was mediated by the alternative pathway of complement.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗
PubMed1994

Staurosporine inhibits invasion of erythrocytes by malarial merozoites.

Staurosporine, a protein kinase inhibitor, inhibits the invasion of rhesus by Plasmodium knowlesi merozoites with an IC50 of 250 nM. The drug exerts its effects primarily on the merozoite, with little or no effect on the erythrocyte. Okadaic acid, an inhibitor of protein phosphatases, can partially abrogate the inhibitory effects of staurosporine. Staurosporine arrests invasion at a step which is ultrastructurally similar to the arrest caused by cytochalasins B and D: the merozoite attaches, apically reorients, and forms a junction with the erythrocyte, but it does not internalize. These results suggest that protein phosphorylation within the merozoite plays an important role in the internalization step of invasion.

Alkaloids↗
PubMed1994

Glycophorin B as an EBA-175 independent Plasmodium falciparum receptor of human erythrocytes.

Invasion of erythrocytes by malaria parasites involves multiple receptor-ligand interactions. To elucidate these pathways, we made use of four parasite clones with differing specificities for invasion, erythrocytes that are mutant for either glycophorin A or B, and enzyme modification of the erythrocyte surface with neuraminidase and trypsin. Neuraminidase alone abolishes invasion of two parasite clones (Dd2, FCR3/A2); these invade after trypsin treatment alone. A third clone (7G8) is unable to invade trypsin-treated erythrocytes. The fourth clone (HB3) can invade after either neuraminidase or trypsin treatment. The receptor for invasion of trypsin-treated erythrocytes was explored in two ways: treatment of trypsin-treated normal cells with neuraminidase, and trypsin treatment of glycophorin B-deficient cells. Both treatments eliminated invasion by all clones, indicating that the trypsin-independent pathway uses sialic acid and glycophorin B. To identify parasite proteins involved in the different pathways, erythrocyte binding assays were performed with soluble parasite proteins from each clone. Based on binding assays using erythrocytes that lack glycophorin A, the parasite protein known as EBA-175 appears to bind predominantly to glycophorin A. In contrast, the glycophorin B pathway does not appear to involve EBA-175, as binding of EBA-175 was similarly reduced to trypsin-treated normal and trypsin-treated glycophorin B-deficient erythrocytes. Thus, the glycophorin B-dependent, sialic acid-dependent invasion of trypsin-treated normal erythrocytes uses a different parasite ligand, indicating two or more sialic-dependent pathways for invasion. Clone 7G8, which cannot invade trypsin-treated erythrocytes, may be missing the ligand for invasion via glycophorin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Gender in medical encounters: an analysis of physician and patient communication in a primary care setting.

The relation of physician and patient gender to verbal and nonverbal communication was examined in 100 routine medical visits. Female physicians conducted longer visits, made more positive statements, made more partnership statements, asked more questions, made more back-channel responses, and smiled and nodded more. Patients made more partnership statements and gave more medical information to female physicians. The combinations of female physician-female patient and female physician-male patient received special attention in planned contrasts. These combinations showed distinctive patterns of physician and patient behavior, especially in nonverbal communication. We discuss the relation of the results to gender differences in nonclinical settings, role strains in medical visits, and current trends in medical education.

Adult↗
PubMed1994

Satisfaction, gender, and communication in medical visits.

The authors conducted two studies of routine medical visits, investigating the relation of physician gender, patient gender, and physician age to patient satisfaction, and the correlations between communication behaviors and satisfaction separately for different combinations of patient and physician gender. Study 1 was based on videotaped visits to a hospital-based internal medicine practice (n = 97 visits). Study 2 was based on audiotaped visits to 11 different community and hospital-based practices in the United States and Canada (n = 524 visits). In both studies, patients examined by younger physicians, especially younger female physicians, reported lower ratings of satisfaction. These findings were true for male and female patients; however, in both studies, the lowest satisfaction in absolute terms was among male patients examined by younger female physicians. The effects were not explained by patient and physician background characteristics or by measured communication during the visit. Correlations between verbal and nonverbal communication and satisfaction for different combinations of physician and patient gender suggested that gender-related values and expectations influence patients' reactions to physicians' behavior. There also was evidence that patient satisfaction is reflected in the patient's affective behavior during the visit.

Age Factors↗
PubMed1994

Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

Animals↗
PubMed1993

A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor.

Plasmodium vivax and P. falciparum are the major causes of human malaria, except in sub-Saharan Africa where people lack the Duffy blood group antigen, the erythrocyte receptor for P. vivax. Duffy negative human erythrocytes are resistant to invasion by P. vivax and the related monkey malaria, P. knowlesi. Several lines of evidence in the present study indicate that the Duffy blood group antigen is the erythrocyte receptor for the chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA). First, IL-8 binds minimally to Duffy negative erythrocytes. Second, a monoclonal antibody to the Duffy blood group antigen blocked binding of IL-8 and other chemokines to Duffy positive erythrocytes. Third, both MGSA and IL-8 blocked the binding of the parasite ligand and the invasion of human erythrocytes by P. knowlesi, suggesting the possibility of receptor blockade for anti-malarial therapy.

Animals↗
PubMed1993

Analysis of sequence diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-1).

Immunization with the first identified Plasmodium falciparum merozoite surface protein (MSP-1) protected monkeys from an otherwise fatal infection. The question of whether the high degree of diversity in MSP-1 among parasite clones will be an impediment to its development as a vaccine candidate needs to be resolved. We have aligned all published sequences, identifying errors, resequencing a portion of one parasite clone, and identifying probable duplicate sequences of four pairs of parasite clones. The sequences are displayed in a fashion that facilitates the study of variation and its potentially diverse origins. The original dimorphic sequences described by Tanabe et al. have been modified to include only common sequences throughout the entire gene. The extension of the dimorphic region to the 5' end of block 3 brings into question the involvement of intragenic crossover as the major mechanism generating allelic diversity. Additional diversity developed from point mutations and recombination in certain regions of the gene. The regions of variability and conservation should serve as a data base for planning vaccine trials.

Alleles↗
PubMed1993

The major merozoite surface protein as a malaria vaccine target.

Experts gathered for two days in the summer of 1992 at the National Institutes of Health and the Walter Reed Army Institute of Research to discuss the potential of a major merozoite surface protein (MSP-1) in malaria vaccine development. The participants came in an exemplary spirit of co-operation, sharing ideas and unpublished data toward the common goal of a malaria vaccine. Their conclusions are presented here by Carter Diggs, Ripley Bollou and Lou Miller.

Journal Article↗
PubMed1993

An approach to development of specific T-lymphocyte lines by use of preprocessed antigens in Plasmodium vinckei vinckei murine malaria.

The development of parasite-specific T-cell lines represents one approach to the potential identification of relevant immunogens in erythrocytic malarial infection. However, the use of parasitized-erythrocyte lysates as antigens inhibits the proliferation of T cells. To circumvent this problem, we preincubated antigen-presenting cells (APCs) from spleens of malaria-naive, BALB/c mice with a Plasmodium vinckei vinckei (hereafter referred to as P. vinckei)-parasitized erythrocyte lysate. APCs were subsequently irradiated and washed prior to being incubated with T lymphocytes from P. vinckei-immune, histocompatible mice. After 8 to 10 cycles of antigenic stimulation and rest, two T-cell lines were analyzed. Both lines were predominantly CD4+. Proliferation assays demonstrated marked lymphocyte blastogenesis to syngeneic but not allogeneic APCs that had preprocessed malarial antigen. Antigen incubated directly with T cells and nonpulsed APCs in vitro did not result in T-cell proliferation. Assays of interleukin-2 (IL-2), IL-4, IL-5, and gamma interferon were compatible with one cell line being predominantly TH1 and the other being TH2. Thus, APCs that have preprocessed malarial antigen and are free of extraneous parasite material induce highly reactive, antigen-specific, major histocompatibility complex-restricted T-cell lines that functionally appear capable of inducing humoral and/or cell-mediated immunity.

Animals↗