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P Lepage

Publications and source records attributed to P Lepage.

At least 91 records · Page 5Linked to original sources
PubMed1991

Perinatal transmission of HIV-1: lack of impact of maternal HIV infection on characteristics of livebirths and on neonatal mortality in Kigali, Rwanda.

We present the baseline results of a prospective cohort study on the perinatal transmission of HIV-1 in Kigali, Rwanda. HIV-1-antibody testing was offered to all women of urban origin delivering a live newborn at the maternity ward of the Centre Hospitalier de Kigali from November 1988 to June 1989; 218 newborns of 215 HIV-positive mothers were matched to 218 newborns of 216 HIV-negative mothers. The matching criteria were maternal age and parity. No differences in socioeconomic characteristics were observed between HIV-positive and HIV-negative women. HIV-positive mothers more frequently reported a history of at least one death of a previously born child (P less than 0.01) and a history of abortion (P less than 0.001). Most of the HIV-positive women were asymptomatic, but 72.4% of them had a CD4; CD8 ratio less than 1 versus 10.1% in the HIV-negative group (P less than 0.001). The frequency of signs and symptoms was not statistically different in the two groups, except for a history of herpes zoster or chronic cough, which was more frequent among HIV-positive women. The rates of prematurity, low birth weight, congenital malformations and neonatal mortality were comparable in the two groups. However, infants of HIV-positive mothers had a mean birth weight 130 g lower than the infants of HIV-negative mothers (P less than 0.01). The impact of maternal HIV-1 infection on the infant seems limited during the neonatal period.

Abortion, Spontaneous↗
PubMed1990

Application of electrospray mass spectrometry to the characterization of recombinant proteins up to 44 kDa.

Mass measurement by electrospray mass spectrometry (ESMS) is used as a rapid preliminary verification of the identity of various recombinant proteins ranging from 7 to 44 kDa with an accuracy of 0.01-0.03%. ESMS not only improves the speed but also the reliability of the protein structure determination when used in conjunction with other methods of protein analysis. Modifications of these large molecules, for example the loss of C-terminal amino acids, N-terminal acetylation, 2-mercaptoethanol addition to a cysteine, and trace formation of a covalent dimer (3%), are easily detected individually or in mixtures by mass measurement using ESMS; feats which would be very difficult to achieve using classical biochemical methods. As little as 1% of several structurally related protein contaminants have been identified in a 15 kDa recombinant protein preparation.

Amino Acid Sequence↗
PubMed1990

Treatment of gonococcal conjunctivitis with a single intramuscular injection of cefotaxime.

Between 25% and 50% of children in African countries are exposed to Neisseria gonorrhoeae at birth and are at risk of developing gonococcal ophthalmia neonatorum. The problem is aggravated by the emergence of penicillinase-producing strains of N. gonorrhoeae (PPNG). Twenty-one children (9 boys, 12 girls, mean age 13 days) presenting with gonococcal conjunctivitis were identified during a 47-month period. Nineteen children received a single im injection of 100 mg/kg cefotaxime (maximum dose 1 g) without topical antibiotic therapy. One neonate with concurrent otitis media was treated with cefotaxime for five days and a 17-month old girl received two im injections because of the severity of her symptoms. Eight patients (38%) had received previous antibiotic therapy (penicillin im and/or tetracycline eye ointment). Twelve isolates of N. gonorrhoeae were found to be PPNG strains. At three to seven day follow-up examination all 21 patients were clinically cured without residual eye damage. Cefotaxime offers an effective first line treatment for gonococcal ophthalmia neonatorum in areas where there is a high prevalence of PPNG strains. The advantages of a single-dose treatment over multidose regimens in terms of compliance and cost are discussed.

Cefotaxime↗
PubMed1990

Multiresistant Salmonella typhimurium systemic infection in Rwanda. Clinical features and treatment with cefotaxime.

Children with multiresistant Salmonella typhimurium (MRST) systemic infections, in total 246, were diagnosed during the study period. Of these, 220 had MRST without metastatic focal infections and 26 had metastatic focal infections (including 12 patients with meningitis). The median age of the children was 10 months. Diarrhoeal disease, measles and severe malnutrition were the most frequent causes of admission. Fever was found in 99% and diarrhoea in 72% of the patients, with respiratory symptoms in 72%. In 199 (81%) of the patients, the MRST infection was considered to be hospital-acquired. Of the 246 children, 159 were treated with cefotaxime. In this group, 16 of 152 patients died (10.5%). However, of the 87 children who did not receive cefotaxime, 64 died (74%). Relapses occurred in 4% of the patients with bacteraemia treated with cefotaxime. Our study confirms the high efficiency of cefotaxime in treating severe systemic infections with MRST.

Adolescent↗
PubMed1990

Increased biological activity of a recombinant factor IX variant carrying alanine at position +1.

In attempts to improve the post-translational modification and processing of recombinant factor IX (FIX) we have altered the cDNA sequence encoding pre-pro-FIX using site-directed mutagenesis and have expressed the variant cDNAs in BHK21 cells using a vaccinia-virus-derived vector. We find that substitution of the tyrosine residue at +1 for an alanine increases the biological activity of the recombinant molecules 2-fold. On the other hand, substitution of the proline at -3 for a valine results in no significant change to the specific activity of the protein. Other alterations to the N-terminus of the FIX proteins, in attempts to mimic other vitamin-K-dependent proteins, result in the failure to produce a secreted polypeptide. N-terminal sequence analysis of purified recombinant molecules reveals a correlation between specific activity and the efficiency of correct pro-sequence cleavage. gamma-Carboxylation analysis of purified recombinant proteins indicates that each molecule including unmutated FIX is completely gamma-carboxylated in this system. Thus the observed increase in biological activity of FIX variants containing an alanine at position +1 is not due to increased gamma-carboxylation but, at least in part, to more efficient pro-peptide cleavage.

Alanine↗
PubMed1989

Isolation from human cerebrospinal fluid of a new insulin-like growth factor-binding protein with a selective affinity for IGF-II.

In biological fluids IGF-I and IGF-II are bound to specific, high-affinity binding protein (BPs). Two human BPs have been isolated, one from serum, which is GH-dependent, the other from amniotic fluid (AF BP), and their cDNAs have recently been cloned. We report here the isolation of another, new species from cerebrospinal fluid (CSF) where this BP predominates. The protein was purified to homogeneity by a four-step procedure: gel filtration, chromatofocusing, hydrophobic-interaction chromatography and reverse-phase chromatography. Thereafter, SDS-polyacrylamide gel electrophoresis gave an Mr of 34,000 (non-reduced), chromatofocusing gave an isoelectric point of 5.0m and its affinity for IGF-II (3 x 10(10) M-1) was 10 times that for IGF-I. The N-terminal amino acid sequence of the first 15 residues determined in a BP preparation from the CSF of children was Leu-Ala-Pro-Gly-(/)-Gly-Gln-Gly-Val-Gln-Ala-Gly-Ala-Pro-Gly. A similar sequence was found for adult CSF, apart from residues 12 and 13 (-Leu-Leu-). These are highly analogous with the sequences starting from residue 69 of the GH-dependent BP, and from residue 61 of the AF BP. The new BP isolated is therefore related to, but distinct from, the other human BPs.

Adult↗
PubMed1989

Isolation of recombinant partial gag gene product p18 (HIV-1Bru) from Escherichia coli.

The membrane-associated structural protein, p18, of the human immunodeficiency virus (HIV-1), has been expressed in Escherichia coli. The recombinant protein was purified by cation-exchange chromatography on S Sepharose followed by cation-exchange high-performance liquid chromatography (HPLC) on Sulfoethyl Aspartamide. The isolation of 28.7 mg of recombinant p18 from 16.71 of cell culture represents an overall yield of ca. 20%. Recombinant p18 was characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, reversed-phase HPLC, amino acid composition and amino acid sequence analysis of the N-terminus. Edman degradation of peptides generated by trypsin or Staphylococcus aureus V8 proteolytic digestion, including the C-terminus, confirmed the amino acid sequence to be that predicted from the cDNA. A C-terminally cleaved form of recombinant p18, p18LM, was separated in the cation-exchange HPLC step and was partially characterized in parallel with the intact molecule. By Western blotting it was shown that recombinant p18 in addition to the cleaved form p18LM is recognized by a monoclonal antibody which was generated against the natural protein from HIV-1.

Amino Acid Sequence↗
PubMed1989

Isolation of recombinant hirudin by preparative high-performance liquid chromatography.

The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described. rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation. Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium. The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases. Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Liquid secondary ion mass spectrometry was used to confirm its primary structure. The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active.

Chromatography, High Pressure Liquid↗
PubMed1989

Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032.

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.

Amino Acid Sequence↗
PubMed1989

Mass spectrometry analyses of recombinant hirudins (7 kDa).

The use of liquid secondary ion mass spectrometry (LSIMS) in the characterization of related recombinant 7-kDa peptides illustrates the adequacy of average mass measurement by scanning at low resolution. The difficulty in using the high-resolution technique in the case of poor LSIMS sensitive peptides is discussed, as well as the fact that it does not give, for these molecular weights, any real advantage. The average (or chemical) molecular weights of three recombinant hirudin molecules, hirudin variant 2 (rHV2, 6892.4 Da), hirudin variant 2-Lys47 (rHV2-Lys47, 6906.5 Da), and hirudin variant 2-Arg47 (rHV2-Arg47, 6934.5 Da), less than or equal to 10 micrograms each, have been measured with an accuracy less than or equal to 0.3 Da in the narrow-scan mode and less than or equal to 0.5 Da (from the protonated molecular ion) in the wide-scan mode within 10-15 min; this allows easy distinction of the three 65 amino acid proteins, which differ by a single amino acid. These three molecules could also be distinguished from one another in a mixture. Mass spectrometry and limited sequence characterization of several minor, similarly isolated peptides identified them to be N-terminal additions and/or C-terminal deletions of rHV2-Lys47. LSIMS analysis is consistent with there being no covalent dimer of rHV2-Lys47 as a narrow scan of the 7-kDa molecular ion cluster at high resolution shows it not to be a doubly charged ion.

Amino Acid Sequence↗
PubMed1989

Insect immunity: isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides.

We have isolated from the hemolymph of immunized larvae of the dipteran insect Phormia terranovae two peptides that are selectively active against Gram-positive bacteria. They are positively charged peptides of 40 residues containing three intramolecular disulfide bridges and differ from one another by only a single amino acid. These peptides are neither functionally nor structurally related to any known insect immune response peptides but show significant homology to microbicidal cationic peptides from mammalian granulocytes (defensins). We propose the name "insect defensins" for these insect antibiotic peptides.

Amino Acid Sequence↗
PubMed1989

Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii.

The strategy chosen for cloning potential vaccine antigens of Toxoplasma gondii was based on the hypothesis that the definitive protection observed in natural infection is due to the presence of encysted bradyzoite forms in host tissues throughout life. The antigens released by the bradyzoites would maintain an immune response against the invading tachyzoites. This led us to identify in tachyzoite in vitro translation products a polypeptide of 24 kDa that is an excreted-secreted antigen (ESA) and is cross-reactive with bradyzoites. In addition, the detection of anti-P24 IgG antibodies is correlated with the chronic infection in man. The gene encoding P24 has been isolated, sequenced, and expressed in Escherichia coli and eukaryotic cells. The recombinant proteins were immunogenic in mice, producing anti-native P23 antibodies. Immunocytochemical analysis located the native antigen in the dense granules of both tachyzoite and bradyzoite forms and showed that it is secreted within host-cell-modified phagosome. Moreover 45Ca2+ labeling as well as regional homologies indicate that this protein has Ca2+-binding properties, suggesting its physiological importance in host-cell invasion. P23 is of diagnostic interest as a marker of chronic toxoplasmosis and is proposed as a vaccine component.

Amino Acid Sequence↗
PubMed1989

Evaluation and simplification of the World Health Organization clinical case definition for paediatric AIDS.

The World Health Organization (WHO) clinical case definition for paediatric AIDS was tested during a 1-month period on 221 consecutive hospitalized children in Kigali, Rwanda. Relevant clinical features not included in the WHO case definition were also evaluated. Thirty-four out of the 221 children (15.4%) were HIV seropositive. Although the specificity of the WHO case definition was high (92%), the sensitivity and the positive predictive value (PPV) were low (41 and 48%, respectively). The following individual signs had a PPV at least equal to the complete WHO case definition: chronic diarrhoea (47%), respiratory distress secondary to lower respiratory tract infection (50%), oral candidiasis (53%), parotitis (67%), generalized lymphadenopathy (88%), and herpes zoster infection (100%). When logistic regression analysis was done on the nine variables included in the WHO case definition, confirmed maternal infection was the best predictive variable for HIV seropositivity in children (P less than 10(-5). We further excluded the serological status of the mother from the analysis and performed a stepwise logistic regression analysis on the 18 clinical signs and symptoms for which information had been collected. Those signs and symptoms contributing the most to the regression were: respiratory distress, chronic diarrhoea and generalized lymphadenopathy. Based on these findings, we propose a simplified clinical case definition for paediatric AIDS in Africa with better sensitivity, specificity and PPV than the WHO case definition. Further work is needed using this approach to develop case definitions useful for epidemiological surveillance and for case management.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Adult and paediatric AIDS patients lacking HIV antibodies detected by enzyme immunoassay in central Africa: further serological studies.

We describe HIV antigen and HIV-1, HIV-2 and human T-cell leukaemia virus type I (HTLV-I) Western blot (WB) results in 20 adults and 12 children with AIDS who were shown to be HIV-1 seronegative by two commercial enzyme immunoassays (EIA), in Kigali, Rwanda, central Africa. These patients represented 3% of adults and 7.4% of children with AIDS observed in Kigali during the study period. Thirteen of the adults and five of the children were HIV-1 WB positive. All patients were HTLV-I WB negative. One adult AIDS patient had a reactive HIV-2 WB; he is the first HIV-2-infected individual to be diagnosed in Rwanda. HIV antigenaemia was demonstrated in only one adult and one child, suggesting that HIV antigenaemia is not frequent in African AIDS patients, even in the case of weak immunologic responses to HIV core proteins. Three adults and four children had none of the serological markers detected. This study showed that HIV EIA negativity occurs infrequently in African patients with AIDS. In such a situation, an attempt to detect HIV antibodies by a more sensitive technique, such as WB, is necessary.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Bacteraemia as predictor of HIV infection in African children.

In Rwanda, both HIV infection and bacteraemia represent major health problems among paediatric populations. We carried out of prospective study of determine if bacteraemia is a marker of HIV infection among ambulatory and hospitalized Rwandese children. All children presenting at the Department of Paediatrics of the Center Hospitalier de Kigali who had their blood cultured during a two-month period were eligible for the study. One hundred and thirty-five children were included in the study. A pathogen was isolated from 36 children (26.7%): S. typhimurium (10 cases), S. enteritidis (6), S. typhi (4), Str. pneumoniae (9). H. influenzae (6) and S. aureus (1). No association was found between bacteraemia and HIV seropositivity when all the children were considered. However, among patients less than 2 years old, bacteraemic subjects were more frequently (p less than 0.05) HIV seropositive (44%) than those with negative blood cultures (19%). Our study shows that in young children in Central Africa, the presence of bacteraemia may be an important marker of HIV seropositivity.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Seronegativity and HIV infection.

An African girl born to an HIV seropositive mother was seropositive during the neonatal period, became seronegative, and was again found to be seropositive at 18 and 20 months of age. We suggest that seronegative children born to seropositive mothers should be followed up for months or even years before HIV infection can be ruled out.

Age Factors↗