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L Pereira

Publications and source records attributed to L Pereira.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1989

Domain structure of herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous and discontinuous residues.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is a multifunctional glycoprotein required for infectivity; it is thought to promote fusion of the viral envelope with the cell membrane and entry of virions into cells. To map the antigenic and functional domains on gB, we constructed amino terminal derivatives lacking the entire carboxyl terminus and internal deletion mutants lacking defined regions of the extracellular and transmembrane domains. Transient expression of the mutants in COS-1 cells revealed that the amino terminal derivatives were released into the medium whereas those with deletions in the extracellular domain were mostly retained within the transfected cells. Analysis of intact gB and the amino terminal derivatives showed that the intact molecule formed dimers whereas the mutant derivatives did not. Reactions of the derivatives with a panel of well-characterized monoclonal antibodies to gB showed that the neutralizing epitopes cluster in two domains. The first maps in the amino terminal 190 residues and contains seven continuous epitopes, five of which are HSV-1-specific. Reactions of antibodies with a set of oligopeptides fine-mapped the epitopes between residues 1 and 47. The second domain is composed of discontinuous epitopes and was expressed by amino terminal derivatives that were at least 457 residues in length or longer. Eleven epitopes map in this region, including those of four potent neutralizing antibodies whose cognitive sites mapped between residues 273 and 298 in mapping studies using antibody-resistant mutants. Results of the present study indicate that the cognitive sites of these antibodies are assembled into the discontinuous domain by juxtaposing residues from the amino-terminal half of gB monomers.

Amino Acid Sequence↗
PubMed1989

A subset of type-specific epitopes map in the amino terminus of herpes simplex virus type 1 glycoprotein B.

We constructed a recombinant herpes simplex virus (HSV) containing the transcribed coding and non-coding sequences of HSV-1 strain F glycoprotein B (gB) gene, a gamma 1 gene, fused to the promoter-regulatory sequences of the HSV-1 alpha 4 gene and inserted into the thymidine kinase gene of RH1G44, an HSV-1 x HSV-2 recombinant that contains an HSV-2 gB gene at the natural locus. Phenotypic analyses of the insertion mutant, R3145, showed that the alpha gB gene was transcribed in the presence of cycloheximide but underwent partial conversion to the HSV-2 form. Nucleotide sequencing of the gene indicated that the 5' crossover occurred between nucleotides 107 and 117 upstream from the translation initiation site and that the 3' crossover occurred between the sequences specifying amino acids 402 and 412 of the HSV-1 gB. The chimeric protein consisted of an N-terminal 405 to 415 amino acids encoded by the HSV-2 gene and a C-terminal 462 to 472 amino acids encoded by the HSV-1 gene. Comparison of the reactivity of the parental and recombinant gB with type-specific monoclonal antibodies indicated that the chimeric gB lost reactivity with four HSV-1-specific antibodies but gained reactivity with three HSV-2-specific antibodies.

Amino Acid Sequence↗
PubMed1989

A major neutralizing domain maps within the carboxyl-terminal half of the cleaved cytomegalovirus B glycoprotein.

Cytomegalovirus (CMV) encodes several glycoproteins reported to be structural homologues of glycoproteins encoded by herpes simplex virus type 1 (HSV-1). To map the antigenic and functional domains on the 907 amino acid CMV glycoprotein B (gB), we cloned and expressed a subfragment of BamHI fragment R of the CMV (Towne) genome into an expression vector and reacted the resulting gene product with a panel of monoclonal antibodies. Our results showed that the DNA fragment encodes related glycoproteins which we previously designated gA and which others have reported to be homologous to HSV-1 gB in CMV (AD169). Analyses of the processing of CMV gB transiently expressed in eukaryotic cells showed that glycosylation occurred independently of viral infection. Ten antibodies with complement-dependent and independent neutralizing activity reacted with a truncated derivative of gB that contained 619 amino-terminal residues but lacked the transmembrane and intracellular regions of the molecule. Twelve additional antibodies reacted with a CHO cell line expressing a 680 amino-terminal derivative of gB. All of the reactive antibodies precipitated the 447 residue carboxy-terminal cleavage product of gB from extracts of CMV-infected cells. These results showed that the neutralizing epitopes map in at least two domains of gB which are located in a discontinuous segment of 219 amino acids between residues 461 and 680 from the amino terminus of the molecule.

Cytomegalovirus↗
PubMed1988

Antibody-resistant mutations in cross-reactive and type-specific epitopes of herpes simplex virus 1 glycoprotein B map in separate domains.

To characterize the domains of HSV-1 glycoprotein B (gB), we isolated mutants resistant to monoclonal antibodies with potent neutralizing activity. Partial nucleotide sequencing of the mutations revealed that gB contains two domains comprising discontinuous and continuous amino acids that bind cross-reactive and type-specific neutralizing antibodies. Four mutations in a discontinuous domain, R1435, R233, R1375, and R126, contained substitutions of Tyr278 for His278, His298 for Arg298, Gln274 for Arg274, and Asn273 for Tyr273, respectively. Two mutations in a continuous domain, R1392 and R1397, contained substitutions of Thr32 for Ala32 and Thr47 for Asn47, respectively, and overlapped two other type-specific epitopes. Analysis of the nucleotide sequence of strain KOS showed differences from strain F at four residues proximal to the R1392 mutation and one residue proximal to the R1397 mutation, which explains the failure of HSV-1(F)-specific antibodies to these epitopes to react with KOS. One target site for proteolytic cleavage of gB by cellular enzymes maps at the amino terminus, partially overlapping four HSV-1-specific epitopes.

Amino Acid Sequence↗
PubMed1988

Characterization of epitopes on native and denatured forms of herpes simplex virus glycoprotein B.

Herpes simplex virus 1 glycoprotein B (gB) is an envelope glycoprotein which promotes fusion of virions with the cell membrane. This report characterizes the epitopes on native, disulfide-linked dimers of gB and monomeric forms of the glycoprotein using a panel of monoclonal antibodies. The antibodies were divided into groups, based on immune reactions with denatured or native forms of gB. The first group reacted with discontinuous epitopes assembled on gB dimers but failed to detect native or denatured monomers. In contrast, the second group reacted with denatured gB recognizing continuous epitopes on the parent oligomer and monomeric forms. Comparison of gB dimers formed by HFEM and tsB5 revealed that mutant forms specified altered immunological properties. Analysis of gB made in Vero cells showed that discontinuous epitopes were retained whereas a subset of continuous ones were lost on the cleavage products. Results of this study indicate that more than half of the epitopes on gB are generated by juxtaposing amino acids from one or more gB subunits and differ from continuous epitopes present on both forms of the molecule.

Animals↗
PubMed1988

Type-specific antibodies to herpes simplex virus type 2 (HSV-2) glycoprotein G in pregnant women, infants exposed to maternal HSV-2 infection at delivery, and infants with neonatal herpes.

We used a murine monoclonal antibody to herpes simplex virus (HSV) type 2 (HSV-2) glycoprotein G (gG) to develop an enzyme immunoassay that detected HSV-2 type-specific antibodies in human sera. Antibodies to HSV-2 gG were detected in 98 (96%) of 102 sera from pregnant women with culture-proved HSV-2 infection. Sixty-five percent of the women had serological evidence of past HSV-2 infection by the Rawls index, based on titers of neutralizing antibody to HSV type 1 and HSV-2. Thirty (88%) of 34 infants exposed to maternal HSV infection at delivery had antibodies to HSV-2 gG and remained well. One infant exposed to primary maternal HSV-2 infection lacked antibodies to HSV-2 gG and developed neonatal HSV-2 infection. The mean +/- SD optical density by HSV-2 gG enzyme-linked immunosorbent assay for sera obtained from 17 infants within one week after onset of neonatal HSV-2 infection was 0.25 +/- 0.12, compared with 1.15 +/- 0.34 in cord blood sera from exposed infants who did not develop symptoms (P less than .0001 by t test).

Antibodies, Viral↗
PubMed1988

Human cytomegalovirus ICP22, the product of the HWLF1 reading frame, is an early nuclear protein that is released from cells.

We employed a murine monoclonal antibody (CH41) and a lambda gt11 library of human cytomegalovirus (CMV) DNA fragments to map the gene for a viral protein, denoted infected cell protein (ICP) 22, to the HWLF1 open reading frame in the S component of the CMV genome (0.92 to 0.93 map units). By using antibody CH41 in immunofluorescence, immunoprecipitation and immunoblotting analyses, ICP22 was readily detected as a beta (delayed early) gene product during viral growth. The cellular localization of this protein was found to be nuclear by immunofluorescence analysis; however, it partitioned with the cytoplasm when cells were fractionated with non-ionic detergents. Analysis of cell-free medium showed that a proportion of ICP22 was released from cells as a soluble protein at both early (24 h) and late (72 to 120h) times in infection. The function of this protein which has such diverse characteristics remains unknown.

Antibodies, Monoclonal↗
PubMed1988

Comparative study on O-linked oligosaccharides of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoproteins D1 (gD1) and D2 (gD2) of herpes simplex virus type 1 and type 2, respectively, were purified from infected HEp-2 cells labelled with [3H]glucosamine for 14 h followed by a 3 h chase using HD1 monoclonal antibody linked to Sepharose. O-linked oligosaccharides were found to be present in both glycoproteins. The identification of N-acetyl [3H]galactosaminitol as the major labelled component in the oligosaccharides generated by mild alkaline borohydride treatment demonstrated that these chains have N-acetylgalactosamine at the reducing end. These oligosaccharides consist of mono- and disialylated species with a predominance of the latter species in gD1. Size analysis and radioactive amino sugar composition strongly suggest a structure in which the galactosyl-N-acetylgalactosamine core is substituted with one or two sialic acid residues. In terms of [3H]glucosamine-derived radioactivity, O-linked oligosaccharides are less represented than N-linked oligosaccharides. The O-linked oligosaccharide number determination showed that gD1 and gD2 carry two and three chains, respectively.

Acetylgalactosamine↗
PubMed1988

Mapping of functional and antigenic domains of the alpha 4 protein of herpes simplex virus 1.

Monoclonal antibodies to alpha 4, the major regulatory protein of herpes simplex virus 1, have been shown to differ in their effects on the binding of the protein to its DNA-binding site in the promoter-regulatory domain of an alpha gene. To map the epitopes, we expressed truncated genes in transient expression systems. All 10 monoclonal antibodies tested reacted with the N-terminal 288-amino-acid polypeptide. To map the epitopes more precisely, 29 15-mer oligopeptides, overlapping by five amino acids at each end, were synthesized and reacted with the monoclonal antibodies. The nine reactive monoclonal antibodies were mapped to seven sites. Of the two monoclonal antibodies which blocked the binding of alpha 4 to DNA, one (H950) reacted with oligopeptide no. 3 near the N terminal of the protein, whereas the second (H942) reacted with oligopeptide no. 23 near the C terminus of the 288-amino-acid polypeptide. In further tests, oligopeptide no. 19 was found to compete with two host proteins, designated as alpha H1 and alpha H2-alpha H3, for binding to DNA as well as to retard DNA in a band shift assay, whereas oligopeptides no. 26, 27, and 28 enhanced the binding of alpha 4 to DNA. Moreover, oligopeptide no. 27 was also found to retard DNA in a band shift assay. Polypeptide no. 19 competed with alpha 4 for binding to DNA, whereas no. 27 neither enhanced nor competed with the binding of the host polypeptide alpha H1 to its binding site in the promoter-regulatory domain of an alpha gene, but did enhance the binding of the alpha H2-alpha H3 protein to its binding site. In contrast to these results, the truncated alpha 4 polypeptide, 825 amino acids long, bound to the viral DNA, whereas a shorter, 519-amino-acid-long, truncated polypeptide did not. The 825-amino-acid polypeptide was previously shown to induce in transient expression of a late (gamma 2) viral gene.

Amino Acid Sequence↗
PubMed1987

Conserved antigenic and functional domains on glycoprotein B of herpes simplex virus 1 and bovine herpesvirus 2. Brief report.

A panel of monoclonal antibodies to glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) reacted in immune precipitation and in immunofluorescence tests with a glycoprotein 130,000 in apparent molecular weight specified by bovine herpesvirus 2 (BHV-2). Two cross reactive antibodies neutralized the heterologous virus. These results suggest that the BHV-2 glycoprotein is the homologue of HSV-1 gB and shares structural and functional domains expressed on the gB gene product.

Antibodies, Monoclonal↗
PubMed1987

Immunization of experimental animals with reconstituted glycoprotein mixtures of herpes simplex virus 1 and 2: protection against challenge with virulent virus.

Artificial mixtures of the glycoproteins B, C, D, and E of herpes simplex virus 1 and 2 (HSV-1 and HSV-2), purified individually from infected Vero cell lysates by immunoaffinity to monoclonal antibodies, were bound to an aluminum hydroxide gel and were used to immunize mice, guinea pigs, and owl monkeys (Aotus trivirgatus) once or twice (mice and guinea pigs) to as many as four times (owl monkeys). In all animals tested, low levels of neutralizing antibodies were detected only after two or more immunizations. Lymphocyte transformation tests in owl monkeys suggested low or borderline levels of cellular immunity. The survival of immunized mice after intracerebral challenge was inversely related to the challenge dose. Immunized guinea pigs challenged by intravaginal inoculation showed reduced morbidity at the site of inoculation and were protected from CNS disease. Both immunized and nonimmunized monkeys were highly susceptible and could not be differentiated with respect to morbidity or mortality when challenged with 1,000 pfu of HSV-2 by the intravaginal route.

Animals↗
PubMed1987

Kinetics of expression of herpes simplex virus type 1-specific glycoprotein species on the surfaces of infected murine, simian, and human cells: flow cytometric analysis.

The kinetics of expression of the herpes simplex virus type 1-encoded major glycoprotein species gB, gC, gD, and gE on the surfaces of cells of murine, simian, and human origins were studied. Viable cells were stained with monoclonal antibodies specific for each species, and the levels expressed were determined by fluorescence flow cytometry. Differences were observed in both the kinetics and the levels of expression of individual glycoprotein species, depending upon the origin of the host cells. Glycoprotein gC was expressed early and at high levels in cells of murine and human origins, but late and at relatively low levels in simian cells. In contrast, gE was expressed at high levels in simian cells, but was not detectable until late in the infectious cycle in murine and human cells. The kinetics and levels of expression of gB were similar for all cells investigated, whereas gD, with high levels of expression in all cells late in infection, appeared on the surfaces of murine cells very early postinfection. This approach has allowed a simple quantitative method for comparing levels of glycoprotein expression.

Animals↗
PubMed1987

A cytomegalovirus protein with properties of herpes simplex virus ICP8: partial purification of the polypeptide and map position of the gene.

We demonstrated the presence of a single-stranded DNA-binding protein in human cytomegalovirus (CMV)-infected cells with properties analogous to those of herpes simplex virus (HSV) ICP8. Using monoclonal antibody specific for the CMV protein, we analyzed its fluorescence pattern and time of synthesis, mapped the gene encoding it by using a lambda gt11 library of CMV DNA fragments, and monitored its purification by phosphocellulose and DNA-Sepharose chromatography. In all characteristics we examined, the CMV protein behaved analogously to HSV ICP8. Our results are consistent with a functional role of CMV ICP8 in viral replication that is similar to that of HSV ICP8 and with the evolutionary conservation of the gene of interest in two divergent herpesviruses.

Amino Acid Sequence↗