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Biomedical subjects

L Pereira

Publications and source records attributed to L Pereira.

At least 19 recordsLinked to original sources
PubMed1992

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.

Cell Survival↗
PubMed1992

Domains of herpes simplex virus I glycoprotein B that function in virus penetration, cell-to-cell spread, and cell fusion.

Herpes simplex virus 1 glycoprotein B (gB) is one of 10 glycoproteins in the virion envelope and in the membranes of infected cells. It is required for infection of cells in culture and functions in penetration of the cell by fusing the virion envelope with the plasma membrane. In studies to map the functional domains on HSV-1 gB, we reported that epitopes of potent neutralizing antibodies cluster in three major antigenic domains, D1, D2, and D5a. D1 contains continuous epitopes in the very amino terminus of gB. D2 comprises discontinuous epitopes that are assembled on gB derivatives 457 amino acids in length. D5a contains discontinuous epitopes that map between amino acids 600 and 690. We have now analyzed the function of these domains in virion infectivity by a detailed examination of the effects of 16 neutralizing antibodies on virion adsorption, penetration, plaque development, and cell fusion. Our results are as follows. (i) Ten antibodies with complement-independent neutralizing activity blocked penetration of virions into cells but not their adsorption to the cell surface. Treating cell-bound, neutralized virus with the fusogenic agent polyethylene glycol promoted their entry into cells. (ii) Ten antibodies with complement-dependent and -independent neutralizing activity interfered with plaque development by preventing spread of virus from infected to neighboring uninfected cells. (iii) Nine neutralizing antibodies, all complement-independent, prevented cell fusion induced by strain HFEM syn. We conclude that domains mapping in three regions of gB function in penetration of virions into cells, and that most neutralizing antibodies to these domains also block cell-to-cell spread of virus and cell fusion. The findings that three complement-independent neutralizing antibodies that blocked penetration did not inhibit plaque development, and that only one of these blocked cell fusion, indicate that the cell-to-cell spread of virus and cell fusion are related processes, but not identical to the penetration function.

Antibodies, Monoclonal↗
PubMed1992

Malaria parasitological indices in the Cordillera Province (Santa Cruz Department, Bolivia).

In 1988-1989, as part of a co-operative programme with the local Unidad Sanitaria, two cross-sectional surveys were carried out to study the prevalence of malaria in eight villages in the rural areas near Camiri, Boyuibe and Gutierrez (Cordillera Province, Santa Cruz Department, Bolivia). Thick and thin blood films were collected from all available two- to nine-year-old children at the end of the dry season, in the first survey (252 samples), and after the rainy season, in the second survey (346 samples). The parasite and gametocyte indices increased between surveys from 1.59-25.72 and from 0.40-1.73, respectively. All infections were due to Plasmodium vivax. Both prevalence and parasite load were lower in children aged two to four years than in older children. Prevalences, parasite loads, and parasite densities were highest in rural areas near Camiri and Gutierrez.

Age Factors↗
PubMed1992

Epitopes of herpes simplex virus type 1 glycoprotein B that bind type-common neutralizing antibodies elicit type-specific antibody-dependent cellular cytotoxicity.

A panel of 45 well-characterized monoclonal antibodies (MAbs) reactive to glycoprotein B (gB) of herpes simplex virus (HSV) type 1 was tested by ELISA and in antiviral functional assays (that included virus neutralization, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated lysis), using type 1 or 2 virus strains. All MAbs were ELISA-reactive. Eleven of the MAbs mediated neutralization and 9 mediated ADCC. All of the ADCC epitopes were contained within the amino-terminal half of the extracellular portion of gB. The ADCC reactions were strictly type 1-specific, whereas 9 of 11 neutralizing MAbs exhibited type-common activity. There was some association between the ADCC and neutralization activities, since of 12 MAbs with functional activity, 8 were positive in both assays. These results suggest that differences in the presentation of gB, and perhaps HSV-1 gB versus HSV-2 gB, on free virus and virus-infected cells determine epitope availability.

Animals↗
PubMed1992

Assembly of conformation-dependent neutralizing domains on glycoprotein B of human cytomegalovirus.

We analysed the antigenic properties of human cytomegalovirus (CMV) glycoprotein B (gB) by constructing a set of deletion derivatives lacking different portions of the carboxy terminus and reacting them with a panel of monoclonal antibodies with neutralizing activity. We found that two novel antigenic domains that bind neutralizing antibodies were assembled on truncated forms of gB, one in the amino-terminal half and one that spans the midregion of the molecule. Assembly of the conformation-dependent epitopes occurred independently of residues in the carboxy-terminal half of the molecule and did not depend on proteolytic cleavage of the molecule between amino acids 460 and 461. Ten antibodies recognized a derivative with 447 amino-terminal residues; their failure to recognize a derivative 411 residues long suggested that the amino acids required for assembly of these epitopes either were incorrectly folded, or had been totally or partially deleted in this derivative. Epitopes for three antibodies with complement-independent neutralizing activity were assembled when amino acids from the midregion of gB between residues 447 and 476 were present. Two other antigenic domains were formed by the addition of residues 476 to 618 and 619 to 645 from the carboxy-terminal half of gB. Our results underscore the importance of conformation in the antigenic structure and functional properties of both the amino- and carboxy-terminal portions of gB.

Animals↗
PubMed1992

The amino terminus of human cytomegalovirus glycoprotein B contains epitopes that vary among strains.

We mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DC1v, maps at the amino-terminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMV-infected cells by flow cytometry with antibodies to the amino- and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.

Amino Acid Sequence↗
PubMed1992

Glycoprotein gp116 of human cytomegalovirus contains epitopes for strain-common and strain-specific antibodies.

Glycoprotein gp116 of human cytomegalovirus (HCMV) is a target for neutralizing antibodies. Gp116 is a component of the gCI complex which consists of gp58 and gp116. Like its homologue, glycoprotein B of herpes simplex virus type 1, gp116 contains a highly antigenic region in the N-terminal part of the molecule, between amino acids 28 and 84. Prokaryotic expression plasmids and synthetic peptides were used to define binding sites for mouse and human monoclonal antibodies (MAbs) as well as HCMV convalescent sera. Site I, located between amino acids 68 and 77, contains an epitope recognized by the human MAb C23, which is capable of neutralizing HCMV independently of complement and the site is conserved between HCMV strains. Of HCMV-positive human sera, 53% recognized site I. Site II was mapped using mouse MAbs as well as human sera. It is located between residues 50 and 54, an area which is not conserved between strains AD169 and Towne, the two laboratory strains of known sequence. Strain-specific antibodies were detected in 25% of human sera. Site II-specific antibodies, purified from human sera by affinity chromatography, were found to be incapable of neutralizing HCMV in tissue culture.

Amino Acid Sequence↗
PubMed1992

The fibroblast growth factor receptor is not required for herpes simplex virus type 1 infection.

The early events mediating herpes simplex virus type 1 (HSV-1) infection include virion attachment to cell surface heparan sulfates and subsequent penetration. Recent evidence has suggested that the high-affinity fibroblast growth factor (FGF) receptor mediates HSV-1 entry. This report presents three lines of experimental evidence showing that the high-affinity FGF receptor is not required for HSV-1 infection. First, rat L6 myoblasts lacking FGF receptors were as susceptible to HSV-1 infection as L6 cells genetically engineered to express the FGF receptor. Second, a soluble FGF receptor fragment that inhibited FGF binding and receptor activation did not inhibit HSV-1 infection. Finally, basic FGF (but not acidic FGF) inhibited HSV-1 infection in L6 cells lacking FGF receptors, presumably by blocking cell surface heparan sulfates also required for HSV-1 infection. These results show that the high-affinity FGF receptor is not required for HSV-1 infection but instead that specific low-affinity basic FGF binding sites are used for HSV-1 infection.

Animals↗
PubMed1992

Herpes simplex virus envelopment and maturation studied by fracture label.

Herpes simplex virus envelopment and maturation were investigated by thin-section fracture label. The distribution of glycoproteins B and D was analyzed by labeling with antibodies; the precursor and mature forms of the glycoproteins were differentiated by labeling with the lectins concanavalin A (ConA) and wheat germ agglutinin (WGA), respectively. We report that the two glycoproteins were readily detected in the intracellular virion, whether located between the inner and outer nuclear membranes or within cytoplasmic membrane-bound vesicles and in the inner and outer nuclear membranes themselves. The enveloped virion between the inner and outer nuclear membranes labeled with ConA but not with WGA. During the transit to the extracellular space the reactivity of the virion membranes with ConA decreased and that with WGA ensued. The results document that herpes simplex viruses acquire at the inner nuclear membrane an envelope carrying the immature forms of the glycoproteins and that during the transit to the extracellular space the envelope glycoproteins become of the fully processed type.

Animals↗
PubMed1992

A quantitative assay of retrograde transported HSV in the trigeminal ganglion.

The relationship between the dose of Herpes simplex virus type 1 (HSV) inoculated in the cornea and the amount of actively replicating virus recovered from mouse trigeminal ganglion cells 5 d after corneal scratch and inoculation was investigated with a tissue culture plaque assay. A dose response curve of productive viral replication was obtained. The estimated dose of HSV that produces half-maximal recovery of virus within the ganglion was 9.15 x 10(3) plaque forming units per eye, and the maximal amount of HSV recovered was 1.34 x 10(4) pfu per ganglion. This definition of infectivity as a function of dose will be useful for studying the effects of potential inhibitors of the binding, uptake, and transport of HSV by productively or latently infected trigeminal neurons.

Animals↗
PubMed1991

Mutations in conformation-dependent domains of herpes simplex virus 1 glycoprotein B affect the antigenic properties, dimerization, and transport of the molecule.

Glycoprotein B (gB) is a component of the herpes simplex virus 1 envelope that is required for penetration of virions into cells. We constructed 11 mutants in the gB gene by deleting the carboxy terminus of the molecule, inserting linkers into the ectodomain and intracellular region, and creating point mutations in cysteine residues. To identify regions of the molecule that affect the formation of epitopes on gB, we cloned the mutated genes into a eukaryotic expression vector, transfected them in COS-1 cells, and reacted the gene products in immunofluorescence and immunoprecipitation tests with a panel of monoclonal antibodies. Our findings are as follows. (i) The ectodomain of gB between residues 600 and 690 is highly antigenic and contains residues that specify 8 continuous epitopes and affect the conformation of 12 discontinuous epitopes. Residues that form a novel neutralizing domain and affect the assembly of gB dimers are contained in this region. Dimerization of gB does not require the transmembrane region or the intracellular carboxy terminus. (ii) Transport of the insertion mutants was aberrant and depended on the site mutagenized. Insertions of linkers at residues 391, 413, and 479 of the ectodomain precluded the binding of neutralizing antibodies that recognize residues in four discontinuous-epitope domains; the latter mutant in intact gB was not translocated to the cell surface. In contrast, insertions at residue 600 of the ectodomain and 810 of the intracellular domain did not affect the conformation-dependent epitopes or gB transport. (iii) Substitution of serines for cysteine residues in a discontinuous-epitope domain in the midregion of gB altered the conformation of both proximal and distal sites. Seven epitopes were lost by mutagenesis of cysteine 382 and 4 epitopes by mutagenesis of cysteine 334. Together with previous findings, these results indicate that the ectodomain of gB contains three topographically distinct neutralizing regions, one of continuous and two of discontinuous epitopes. The continuous-epitope domains that map at the amino terminus are not altered by distal mutations. In contrast, the domains of discontinuous epitopes, assembled by juxtaposing residues on the surface of gB, are affected by proximal and distal mutations that alter the antigenic structure, processing, and surface transport of gB.

Amino Acid Sequence↗
PubMed1991

A mutation in the ectodomain of herpes simplex virus 1 glycoprotein B causes defective processing and retention in the endoplasmic reticulum.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is one of several envelope glycoproteins required for virion infectivity and is the only one known to oligomerize into homodimers. To study the conformational constraints for translocation of HSV-1 gB to the surface of eukaryotic cells, we analyzed the transport through the exocytic pathway of the wild-type glycoprotein and of mutant forms with insertions in the ectodomain and intracellular carboxy terminus. Transient expression of the glycoproteins in COS-1 cells showed that an insertion at position 479 in the amino-terminal ectodomain of gB, shown previously by reactions with monoclonal antibodies to have altered the conformation of the molecule, also had a drastic effect on transport, precluding exit of the mutant from the endoplasmic reticulum (ER) and transport to the Golgi and the plasma membrane. The fact that the mutant, gB-(Lk479), formed dimers suggests that local changes in assembled regions caused the transport defect. Mutants containing insertions at residues 600 of the ectodomain and 810 in the intracellular domain were slightly retarded in their rate of transport from the ER to the Golgi. The glucose-regulated proteins GRP78 and GRP94, which are resident proteins of the ER, associated with partially glycosylated, faster-migrating forms of gB but not with the fully processed, more slowly migrating product. GRP78 and GRP94 formed complexes with the mutant gB-(Lk479), which was degraded in the ER. Our results indicate that GRP78, and perhaps also GRP94, acts as a chaperone in the assembly of native gB oligomers and also binds to aberrant forms of the molecule, arresting their transport from the ER and possibly serving as markers for protein degradation in this compartment of the exocytic pathway.

Animals↗
PubMed1991

Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I.

The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) protein that can form stable associations with a variety of proteins retained in the ER because of underglycosylation or other conformational changes. In this study, we provide evidence at the transcriptional level that a conformationally abnormal protein, an altered herpes simplex virus type 1 envelope protein that is retained in the ER of a mammalian cell line, transactivates the grp78 promoter. In contrast, the normal viral envelope glycoprotein does not elevate grp78 promoter activity. Using a series of 5' deletions, linker-scanning, and internal deletion mutations spanning a 100-bp region from -179 to -80, we correlate the cis-acting regulatory elements mediating the activation of grp78 by malfolded proteins, glycosylation block, and the calcium ionophore A23187. We show that they all act through the same control elements, suggesting that they share a common signal. We report here that the highly conserved grp element, while important for basal level and induced grp78 expression, is functionally redundant. The single most important element, by linker-scanning analysis, is a 10-bp region that contains a CCAAT motif. It alone is not sufficient for promoter activity, but a 40-bp region (-129 to -90) that contains this motif is essential for mediating basal level and stress inducibility of the grp78 promoter. We show that the transcription factor CTF/NF-I is able to transactivate the grp78 promoter through interaction with this CCAAT motif.

Animals↗
PubMed1991

Induction of HLA-DR expression on thyroid follicular cells by cytomegalovirus infection in vitro. Evidence for a dual mechanism of induction.

Cytomegalovirus (CMV) infection of primary cultures established from human thyroid nodular and normal (paranodular) tissues resulted in induction of human leukocyte antigen (HLA) DR expression on thyroid follicular cells (TFC), as detected by cell-surface immunofluorescence staining with monoclonal antibodies (MAb). Two distinct modalities of induction were observed. The first type occurred in cultures of normal tissue obtained from CMV-seropositive but not seronegative donors, was detected on 30% to 50% of the TFCs, even though the vast majority of these cells failed to show any morphologic or antigenic evidence of individual CMV infection, and was associated with production of gamma-interferon (gamma-IFN) in vitro. The induced molecules displayed the characteristic DR polypeptide profile on immunoprecipitation and electrophoretic analysis. These results demonstrate that CMV infection of normal thyroid cultures may induce DR expression on TFCs in the absence of pre-existing lymphoid infiltrates and suggest that the induction is the result of an in vitro response to CMV by previously sensitized immunocompetent cells present in these primary cultures. Such a response, associated with the release of gamma-IFN, would induce DR expression on neighboring uninfected cells. The second mode of induction occurred in all CMV-infected cultures, regardless of their tissue origin (nodular or normal) or the serologic status of the donors. Up to 50% of infected TFCs at a late stage of infection, having fully developed CMV antigen-positive intranuclear inclusions, also displayed the cell-surface DR-related determinant recognized by one of the four anti-DR MAbs used. This induction was restricted to TFCs, while CMV-infected fibroblastoid cells present in the monolayers were invariably negative. Induction by CMV of major histocompatibility class II antigens on human epithelial cells may have significant implications in the development of normal immune responses against local viral infection, the enhancement of alloimmune rejection of grafted organs, and the generation of organ-specific autoimmune responses.

Adult↗
PubMed1990

Analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein D and monoclonal antibodies to glycoprotein B.

The role of antibody in neonatal herpes simplex virus (HSV) infection remains controversial. A battery of well-characterized monoclonal antibodies to HSV glycoprotein B (gB), and polyclonal antibodies against synthetic peptides of predicted epitopes of HSV glycoprotein D (gD) were used to determine in vitro functional activity and association with protection against lethal infection in a murine model of neonatal HSV disease. Antiviral neutralization activity of HSV was not associated with antibody-dependent cellular cytotoxicity (ADCC) activity to HSV-infected cells in vitro. In a model of high dose challenge (10(4) PFU), protection was not afforded by any antibody alone, but was by antibody plus human mononuclear cells, and highly associated with ADCC functional activity (P less than 0.001). In a low dose challenge model, neutralizing activity of antibody alone was associated with protection in vivo (P less than 0.001). Of the nine neutralizing epitopes of gD in vitro, eight were predicted surface regions. Four of the five epitopic sites of gD (2-21, 267-276, 288-297, and 303-312) that were determined to be important targets of ADCC and in vivo protection were also predicted to be surface regions. The only exception was the antiserum to region 52-61 which was predicted to be buried and also showed these activities. ADCC as well as neutralizing antibody activity are important in protection against neonatal HSV infection.

Animals↗
PubMed1990

[Iron deficiency in an area of Bolivia and high prevalence of intestinal helminthiasis].

Between November and December 1987, 201 subjects from three communities of the Cordillera Province, Santa Cruz Department, South-East Bolivia (Camiri, Boyuibe, and Gutierrez) were examined for serum iron levels. Sixty-three (31.3%) were iron deficient. There were no significant differences between males and females (37.2% and 26.9%, respectively) and among the three localities studied. Iron deficiency was highest in the 3- to 9-year-old age group and lowest in the 30- to 39-year-old group. Considering the prevalence in the same localities of infections by hookworms, Trichuris trichiura, Strongyloides stercoralis, and Trichostrongylus, it is suggested that hookworms and possibly other intestinal parasites may be an important cause of the observed iron deficiency.

Adolescent↗