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L H Perrin

Publications and source records attributed to L H Perrin.

At least 19 recordsLinked to original sources
PubMed1992

Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite.

Multiplication of the human malaria parasite Plasmodium falciparum within red blood cells is an energy-dependent process and glucose consumption increases dramatically in infected red blood cells (IRBC) versus normal red blood cells (NRBC). The major pathway for glucose metabolism in P. falciparum IRBC is anaerobic glycolysis. Phosphoglycerate kinase (PGK) is one of the key enzymes of this pathway as it generates ATP. We found that the PGK specific activity in P. falciparum IRBC is seven times higher than that in NRBC. The parasitic origin of the increase in PGK activity is confirmed by isoelectric focusing. Indeed, two P. falciparum isoenzymes with neutral isoelectric points were detected. P. falciparum PGK in purified form has a molecular mass of 48 kDa. Antiserum raised against purified P. falciparum PGK specifically recognizes the 48-kDa protein band in P. falciparum and also reacts with P. berghei and P. yoelii IRBC lysates but does not cross-react with PGK associated with NRBC.

Animals↗
PubMed1992

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.

Animals↗
PubMed1992

Quantitation of human immunodeficiency virus provirus and circulating virus: relationship with immunologic parameters.

Virologic and seroimmunologic parameters were determined in 56 persons infected with human immunodeficiency virus (HIV). The provirus level varied from 10 to 100,000/10(6) CD4+ lymphocytes, and genomic HIV RNA was detectable in 39 of 56 patients at a relative concentration varying from 10 to greater than 250 copies/mL of serum. Provirus expressed as copies per 10(6) CD4+ lymphocytes and as circulating virus per milliliter of serum increased with disease progression and decrease of CD4+ cell concentration. The mean provirus concentration expressed per milliliter of blood varied little among categories of patients with various levels of CD4+ cells, but there was a progressive increase of circulating HIV genomic RNA. These virologic data suggest that during the course of HIV infection, an increasing proportion of the remaining CD4+ lymphocytes harbor the HIV genome and produce infectious virus. Finally, there was a marked correlation between increased provirus and genomic RNA concentration and three seroimmunologic markers: decrease in CD4+ cell count, p24 antigenemia, and disappearance of antibodies to HIV core antigen.

Base Sequence↗
PubMed1992

Frequencies of HIV-reactive B cells in seropositive and seronegative individuals.

Peripheral blood mononuclear cells (PBMC) from HIV-infected seropositive (HIV+) but not from normal, seronegative (HIV-) individuals are known to produce anti-HIV antibodies in vitro, in the absence or presence of pokeweed mitogen (PWM). Previous studies showed that up to 20-40% of spontaneously immunoglobulin-secreting B cells from HIV+ individuals are HIV-specific. To analyse the frequency of anti-HIV B cells among 'total' peripheral blood B cells in the present study, we used a limiting dilution assay in which EL-4 thymoma cells induce clones of immunoglobulin-secreting cells in activated as well as resting B cells. Anti-HIV B cells were detected not only in 11/12 HIV+ individuals (with frequencies from 1/910 to 1/21,500 B cells cultured; one negative test was from a person undergoing seroconversion), but also in 4/9 HIV- normal blood donors (1/16,200 to 1/49,000 B cells cultured) and in 3/6 newborns from HIV- mothers (1/11,800 to 1/26,600 B cells cultured). The mean frequency was nine times higher in the HIV+ individuals than in the normal donors. As in previous studies, only the cells from HIV+ individuals generated anti-HIV antibodies in PBMC bulk cultures with or without PWM. The relative proportion of specific anti-HIV antibody/total immunoglobulin in PBMC bulk cultures was 800 times higher by the mean than in EL-4 B cell cultures from HIV+ individuals (whereby the total immunoglobulin secretion for equal numbers of B cells cultured was 500 times lower for PBMC). These different results obtained with different assays suggest that in seropositives most anti-HIV B cells belong to an activated B compartment which is quite small, even in a disease with B cell hyperactivity. Therefore, the specific B cells are strongly diluted among the EL-4 cell-responsive, total B cells. On the other hand, the EL-4 assay can detect HIV-reactive B cells in the B cell repertoire of normal, non-infected individuals.

B-Lymphocytes↗
PubMed1991

Absence of chronic human immunodeficiency virus infection without seroconversion in intravenous drug users: a prospective and retrospective study.

It has been reported that human immunodeficiency virus type 1 (HIV-1) infection may exist in persons without specific antibodies for years. To measure the frequency of a silent carrier state, a study was conducted in a cohort of 124 intravenous drug users (IVDUs) without anti-HIV-1 antibodies. All the participants had engaged in high-risk behavior for HIV-1 transmission for a number of years until 1987 or later. Samples were analyzed at 6-month intervals for the presence of HIV-1 provirus using DNA amplification and for the appearance of anti-HIV-1 antibodies. HIV-1 provirus and antibodies were undetectable in 122 participants, whereas seroconversion was observed in 2. In one of these, both amplified HIV-1 pol gene segment and anti-HIV-1 antibodies were detected simultaneously, and in the other, provirus was detected 1 month before seroconversion. This study suggests that long-term HIV-1 infection without anti-HIV-1 antibodies is rare and that repeated antibody testing is sufficient to determine the HIV-1 status of a person no longer at high risk for HIV-1 infection.

Adult↗
PubMed1991

Comparative evaluation of an ELISA based on recombinant polypeptides and IFA for serology of malaria.

In the present investigation we compare the performance of a solid-phase assay based on three recombinant polypeptides corresponding to three asexual blood-stage antigens of P. falciparum (ELISA MIXT) with the reference method for the measurement of antimalaria antibodies: indirect immunofluorescence antibody assay (IFA). Sera collected from persons with various degrees of exposure to malaria were selected: sera from inhabitants of a malaria endemic area (Group I), European patients with acute malaria infection (Group II) and blood donors with clinical symptoms of sickness or fever during a stay in malaria endemic areas. 86% of the sera gave concording results by ELISA MIXT and IFA. The correlation was 100% for sera of Group I but discrepancies were observed for Groups II and III. The great majority of the differences were due to sera positive on ELISA MIXT but not by IFA. Most of the sera positive on ELISA MIXT reacted with parasite-derived components only on Western-blot. These results underline the potential of the ELISA MIXT for epidemiologic studies.

Adolescent↗
PubMed1990

Human immunodeficiency virus DNA amplification and serology in blood donors.

The significance of indeterminate screening antibody test for human immunodeficiency virus (HIV) serology is still difficult to evaluate, especially in low-risk populations. One hundred twenty-seven blood donors with an initially reactive screening test for HIV antibodies were enrolled in this study. The sera of 95 of these blood donors were reactive on repetition of the test, and none had detectable circulating p24 antigen. Western blot (WB) analysis of the repeatedly reactive sera was as follows: 9 positive, 31 indeterminate, and 55 negative. One of the blood donors with indeterminate WB later presented a seroconversion. On subsequent control 3 to 12 months later, the sera from donors with indeterminate or negative WB did not present any parameters that may indicate a seroconversion. DNA was purified from citrated blood collected from the 127 blood donors at the time of the initial antibody screening. Five micrograms of each DNA sample corresponding to 7 x 10(5) nucleated white blood cells was amplified by polymerase chain reaction (PCR) in the presence of oligonucleotides (primers) corresponding to a highly conserved segment of the pol gene. The detection of amplified DNA was achieved by dot blot and Southern blot using appropriate 32P-labeled oligonucleotides. Ten DNA samples were positive, 9 corresponded to blood donors with a positive HIV serology, and 1 to the blood donor who later presented a seroconversion. These results confirm the sensitivity of the PCR for the diagnosis of HIV infection; they also suggest that repetition of the serology at 3- to 12-month intervals is a valuable procedure for the control of HIV infection status in blood donors.

Base Sequence↗
PubMed1990

Specificity and inhibitory activity of antibodies to Plasmodium falciparum aldolase.

The multiplication of Plasmodium falciparum within RBC is energy-dependent and the glucose consumption of infected RBC is increased more than 50 times over the consumption of normal RBC. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase (p41) have been detected in infected RBC. Expression of the cloned aldolase gene of P. falciparum in Escherichia coli resulted in an enzymatically active polypeptide with a high sp. act. and the recombinant p41 aldolase was used for enzymatic and immunologic studies reported here. The presence of antibodies against p41 in the sera of human adults partially immune to malaria and immunization experiments in monkeys suggest that p41 is implicated in protective immune response against the parasite. Therefore, we analyzed the capacity of various antisera to inhibit P. falciparum aldolase activity. It was found that anti-p41 antibodies raised in mice, rabbits, and monkeys inhibited very efficiently aldolase activity in vitro up to dilutions higher than 10(-3). In contrast none of the human sera with high levels of anti-p41 antibodies were able to inhibit parasite aldolase activity even at a dilution of 1/2. The inability of human antisera to neutralize parasite aldolase is not related to antibody titers but is probably related to the specificity of the human antibodies. This finding is discussed in relation to homology of structure of P. falciparum and mammalian aldolase and to a possible mechanism of parasite adaptation and survival in its natural host.

Animals↗
PubMed1990

Structural diversity of Plasmodium falciparum gp200 is detected by T cells.

T lymphocyte clones (TLC) specific for P. falciparum gp200 (a glycoprotein precursor of the main merozoite surface component) were obtained from two individuals with past exposure to malaria. The 25 established TLC carried the CD4 antigen and proliferated in the presence of immunopurified gp200, crude lysate of the parasite and intact infected red blood cells. They were further tested in proliferation assays for their capacity to recognize the structural diversity displayed by gp200. The stimulating antigen used in these assays was either sonicated or viable preparations of schizonts from five P. falciparum isolates differing in their gp200. The majority of the TLC proliferated similarly in the presence of each of the isolates. One third of the TLC proliferated to a different extent depending on the isolate used for stimulation, while two clones gave isolate-specific responses. These results indicate that the majority of human TLC raised in vitro against gp200, is directed against common determinants. This also suggests that immunization with full length gp200 will not lead predominantly to T cell help restricted to isolate-specific determinant.

Animals↗
PubMed1990

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.

Amino Acid Sequence↗
PubMed1990

Development of a haptoglobin ELISA. Its use as an indicator for malaria.

Hypo- or ahaptoglobinemia, a common phenomenon in tropical countries, is mainly due to malaria induced hemolysis. We have recently shown that the prevalence of hypohaptoglobinemia is a useful epidemiologic indicator in malaria endemic areas. The main limitation to the practical utilisation of this indicator is the available methodologies. Here we describe a sandwich ELISA for the determination of haptoglobin concentrations which is easy to perform, sensitive and requires a minimal amount of equipment. With this test system it is possible to detect haptoglobin in serum at a lower limit of 100 micrograms/ml for 10(-4) dilutions and of less than 1 micrograms/ml for 10(-2) dilutions. This represents at least 200 fold increase in sensitivity as compared to radial immunodiffusion.

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase.

The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.

Amino Acid Sequence↗
PubMed1989

Asexual blood stage vaccines: from merozoites to peptides.

Asexual blood stage proliferation is responsible for the morbidity and mortality associated with malaria infection in man. These developmental stages are therefore obvious targets for the development of malaria vaccines. Several asexual blood stage components have been identified as potential candidates for the development of vaccines and some of them have been shown, following immunization, to induce at least partial protection in a variety of Plasmodium-host combinations. Studies on defined parasite components and on synthetic peptides derived from them have revealed new insights at the molecular level into parasite mechanisms involved in propagation and survival in the infected host, and into the interaction between parasite components and the host immune system. Practical application of these findings is likely to provide the basis for the design of more appropriate antigens for the development of vaccines.

Animals↗
PubMed1988

Variation in p126, a parasitophorous vacuole antigen of Plasmodium falciparum.

We describe a cDNA clone expressing part of p126, a parasitophorous vacuole antigen of Plasmodium falciparum that is processed to smaller fragments about the time of schizont rupture. The amino acid sequence includes determinants that are antigenic during the course of infection. The sequence contains a string of serine residues but no other repetitive elements. Comparison of the sequence to a previously published sequence demonstrates conserved and variable sequence elements. The genomic context of the single copy gene is conserved in six different isolates. p126 is shown to be identical to Pf140, an antigen previously demonstrated to partially protect Saimiri monkeys against P. falciparum infection.

Amino Acid Sequence↗
PubMed1979

[Demonstration of soluble parasite antigens, the corresponding antibodies and immune complexes in acute malaria].

Serum levels of malaria antigens, antimalaria antibodies, immune complexes and complement components have been followed up in 23 patients suffering from acute malaria infection and recovering under therapy. Malarial antigens in serum were detected by counterimmunoelectrophoresis: the peak was observed before therapy started and their levels rapidly decreased. Specific antimalarial antibodies became detectable 5--7 days after starting treatment in patients with a first infection. Immune complexes were detected in 21 of 23 sera with a peak level between days 5 and 9. A marked decrease of C4 and C3 was observed in the presence of normal levels of factor B.

Antibodies↗