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Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes.

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

Animals↗

A vertically transmitted HIV-1 gag-subtype variant detected in Taiwan.

We used polymerase chain reaction (PCR) to amplify a segment, about 560 base pairs (bp), of HIV-1 gag DNA prepared from peripheral blood mononuclear cells of a seropositive Taiwanese pair of mother and infant. TM-1 and TC-1 clones of PCR-amplified DNA derived from the mother and infant, respectively, showed a 94.5% homology with each other. However, the TM-1 and TC-1 sequences exhibited lower degrees of homology, i.e. only 85.1% and 85.8%, respectively, with the corresponding gag segment of a North American HIV-1 subtype (HXB2), and 86.4% and 87.0%, respectively, with that of a Zairean HIV-1 subtype (Z2Z6). The divergence of TM-1 and TC-1 sequences from those of HXB2 and Z2Z6 is particularly prominent in the first (5' proximal) 200 bp of the cloned DNA segment, involving transitions more frequently than transversions. Two additional clones TM-2 and TC-2 derived from the mother and infant were sequenced for the first 200 bp. These four clones showed a high degree of homology (94.7-97.5%) among themselves, providing an evidence for transmission of the virus from the mother to the infant. These findings show the epidemiological value of PCR, and indicate the presence of a gag subtype of HIV-1 which is distinct from both the North American and Zairean subtypes according to the phylogenetic tree constructed.

Adult↗

[Nucleotide sequence of gag and env genes of human immunodeficiency virus type 1, found in Russia: detection of new recombinant variants].

Phylogenetic analysis of nucleotide sequences of gag and env genes of type 1 human immunodeficiency virus (HIV-1) variants isolated from individuals infected through sexual intercourse or nosocomially (by injections with nonsterile syringes) showed that 5 of 27 (18.5%) isolated strains were recombinants. Two viruses found in the Russian Far East had gagAenvE genotype, three other recombinants had envG genotype; gag gene of one isolate belonged to subtype A and gag genes of two others belonged to subtype D. Detection of new recombinant variants in addition to the A/B recombinant described previously shows that these viruses can contribute to the HIV-1 genetic variability in Russia.

Female↗

[The immune response in mice inoculated with HIV-1 gag-gp120 chimeric gene DNA vaccine and IL-18 plasmid].

AIM: To explore the immune response in mice inoculated with HIV-1 chimeric gene gag-gp120 DNA vaccine and IL-18 plasmid. METHODS: BALB/c mice were injected i.m with eukaryotic expression plasmid pVAXIL18 containing IL-18 gene and DNA vaccine plasmid pVAXGE containing HIV-1 gag-gp120 chimeric gene. The specific killing activities of spleen CTL and the titers of serum antibodies of the immunized mice were detected. RESULTS: The specific killing activities of spleen CTL and the titers of serum antibodies in the co-inoculation group were significantly higher than those in the single inoculation group(P<0.05), the vector control group (P<0.01) and PBS control group (P<0.01). CONCLUSION: The specific cellular and humoral immunity in mice can be induced by co-inoculating HIV-1 gag-gp120 chimeric gene DNA vaccine and IL-18 plasmid indicating that IL-18 augments immune response as immunoadjuvant.

Adjuvants, Immunologic↗

[Gagging during oral health care treatments. Available diagnostic and treatment tools].

The aim of this paper was to explore the diagnostic and treatment tools for patients who gag during oral health care treatment. Reference to such tools is not available in scientific literature. For diagnostic purposes, a questionnaire was developed. In due course the results of its reliability and validity will be published. As yet there is no preferred method of dealing with the problem of patients' gagging. A recommended option to treat the problem is based on a cognitive behavioural perspective with gradual exposure in vivo. In cases of extreme gagging, teamwork of oral health care provider and psychologist seems to be a prerequisite for success.

Dental Care↗

Six amino acids from the retroviral gene gag greatly enhance the transforming potential of the oncogene v-erb-B.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds and transforms the corresponding cells in culture. Neoplastic transformation by AEV is mediated principally by an oncogene known as v-erb-B. We have explored the means by which this gene is expressed from the genome of AEV and uncovered an important structural determinant for the potency of the oncogene. In order to define the boundaries of v-erb-B and the supplementary oncogene, v-erb-A, we sequenced all but a small portion of the genome of the ES4 strain of AEV. We then demonstrated that, during expression in infected cells, splicing fuses the first six amino acids of the retroviral gene gag to the body of the v-erb-B protein. In order to explore the impact of this fusion on the function of v-erb-B, we constructed vectors with Murine Leukemia Virus that express the oncogene either with or without the fusion to gag. Viruses generated from these two vectors differed greatly in their abilities to transform cells: fusion of v-erb-B with gag enhanced its transforming ability 50 to 100-fold as determined by focus transformation assays and growth in soft agar. Our data suggest that the difference in transforming ability is not due to alterations in transcription or translation but, rather, may result from changes in post-translational modification.

Amino Acid Sequence↗

The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques.

The simian immunodeficiency virus of macaques (SIVmac) is a lentivirus which induces an AIDS-like disease in rhesus monkeys. We have explored the virus-specific cellular immune response in SIVmac-infected rhesus monkeys. Con A-activated, IL-2 expanded PBL of some SIVmac-infected rhesus monkeys lyse autologous B lymphoblastoid cell lines infected with a recombinant vaccinia virus that carries the SIVmac gag gene. This lysis is mediated by CD8+ lymphocytes and is MHC class I restricted. Moreover, these effector lymphocytes do not express the NK cell-associated molecules NKH1 or CD16. These cells are, therefore, CTL. In a limited prospective study of SIVmac-infected rhesus monkeys, the presence of the SIVmac gag-specific CTL activity in PBL correlated with both a reduced efficiency in isolating SIVmac from PBL of these monkeys and their extended survival. This method for assessing SIVmac gag-specific cellular immunity in rhesus monkeys will be important not only in investigating the immunopathogenesis of SIVmac-induced disease, but also in evaluating the capacity of candidate AIDS vaccines to elicit a cell-mediated immune response in this animal model.

Animals↗

Recognition of human T cell leukemia virus type I (HTLV-I) gag and pX gene products by MHC-restricted cytotoxic T lymphocytes induced in rats against syngeneic HTLV-I-infected cells.

We established rat T cell lines expressing human T cell leukemia virus type I (HTLV-I) Ag from inbred strains of rats, WKA/H, DA, and F344, to study CTL response against the HTLV-I-infected cells. HTLV-I-specific Ag expressed in these rat cells were HTLV-I gag Ag, p19, p24, and p15, and pX Ag, p40tax and p27rex, but not env Ag, as determined by immunofluorescence and immunoblot assays. By immunization of rats with syngeneic HTLV-I-infected cells, CTL against syngeneic HTLV-I-infected cells and antibodies to HTLV-I Ag were generated in WKA/H and DA rats. The bulk CTL cultures from WKA/H and DA rats lysed specifically syngeneic SV40-transformed kidney cells infected with recombinant vaccinia viruses (RVV) expressing HTLV-I gag and pX Ag, but not those infected with RVV expressing HTLV-I env Ag or a control vaccinia virus. From WKA/H rat CTL cultures, four CTL clones reactive with syngeneic HTLV-I-infected cells were isolated, three of which were specific for p27rex/p21x, but the Ag recognized by the other CTL clone was not defined with any RVV used. These results indicate that HTLV-I gag and pX gene products are recognized by MHC-restricted rat CTL specific for syngeneic HTLV-I-infected cells.

Animals↗

An experimental study of the control of the gag reflex with nitrous oxide.

Gagging represents a management problem during dental procedures. A controlled, double blind experiment on human volunteers evaluated the efficacy of nitrous oxide for suppressing experimentally-induced gagging. The ability of the subjects to tolerate palatal and oropharyngeal stimulation was evaluated by measuring the distance of the anatomic palatal and oropharyngeal structure which produce gagging. It was observed that under N(2)O/O(2) inhalation subjects tolerated a significantly more intrusive (deeper) oropharyngeal stimulation than under control conditions.

Adult↗

[Gagging as a symptom of induced anxiety reactions].

In the light of personal studies and relevant reports in the literature, the authors deal with gagging during dental treatment and after denture insertion. In this connection, aetiopathologic and therapeutic aspects of the gagging syndrome are analysed. It is postulated that the gagging reaction is a conditioned anxiety reaction.

Anxiety↗

CTL responses to the gag polyprotein encoded by the murine AIDS defective retrovirus are strain dependent.

Murine AIDS (MAIDS) is induced by a mixture of retroviruses, of which a replication defective virus is the proximal agent of disease. This defective virus harbors a single intact gene that encodes an aberrant gag polyprotein. Certain mouse strains are genetically resistant to MAIDS, with several genes, including H-2Dd, contributing to this resistance. Because MHC class I gene products present intracellular Ags to CTL, recombinant viruses were used to determine whether gag-specific CTLs mediate the genetic linkage between H-2Dd and resistance. Interestingly, while genetically resistant BALB/cByJ and C57BL/KsJ mice (H-2d) generated gag-specific CD8+ CTLs, a similar response was not detected in susceptible BALB.B and C57BL/6J mice (H-2b). However, this CTL response does not appear to be responsible for genetic resistance because: 1) a vigorous CTL response could also be generated by susceptible (C57BL/6 x BALB/cBy) F1 mice and 2) the relevant epitope is H-2Kd restricted.

Animals↗

[The isolation and characteristics of monoclonal antibodies to recombinant proteins of HIV-1 and HIV-2--the env and gag gene products].

Ten hybridomas secreting monoclonal antibodies (Mab) against recombinant HIV-1 and HIV-2 antigens were produced (3 Mab anti-gag protein, 2 anti-env1, and 5 anti-env2). In the immunoblotting assay all the anti-gag Mabs reacted with HIV capsid protein p24, whereas one of them reacted also with p55 protein and with 7 other polypeptides. Another anti-gag Mab cross-reacted with the antigen of subpopulation of human peripheral blood lymphocytes. The third one interacted with the antigen of both HIV-1 and HIV-2. All the 10 Mabs interacted with natural HIV antigens and can be used for identification and differentiation of HIV-1 and HIV-2.

Animals↗

The Gag-Myb-Ets fusion oncogene alters the apoptotic response and growth factor dependence of interleukin-3 dependent murine cells.

Expression of the avian E26-derived Gag-Myb-Ets fusion oncogene in interleukin-3(IL3)-dependent murine hematopoietic cell lines results in a pattern of cell line dependent changes in growth factor-induced proliferation and apoptosis. A drug-selectable retrovirus expressing p135Gag-Myb-Ets induced an erythropoietin(Epo)-responsive phenotype in the cell lines FDC-P2, BaF3 and 32Dc123. Gag-Myb-Ets expression alone did not increase expression of GATA-1 or the Epo receptor(EpoR) in the presence of IL3, and infected cell lines express increased GATA-1 and EpoR only when IL3 was replaced by Epo in the culture media. Indicative of Epo-induced erythroid differentiation, these cells also began to express beta-globin after 3-5 days growth in Epo. Unlike control cells, infected FDC-P2 cells failed to undergo programmed cell death (apoptosis) when transferred from IL3- to Epo-containing media, although a fraction of the cells failed to proliferate following the media shift. Three other IL3-dependent cell lines showed no changes in growth behavior when induced to express the fusion oncogene. Our data shows that Gag-Myb-Ets can have different affects on growth factor pathways depending on the cell background, suggesting a model in which the p135gag-myb-ets fusion oncogene promotes these different responses through its affect on apoptosis.

3T3 Cells↗

The effect of visco-anaesthetic medicament on tactile gag reflex control.

Visco-anaesthetic medication has been used for controlling the gagging sensation in 35 dental patients during intra-oral radiography and impression making. The drug was satisfactory in 29 patients and failed to control the gagging reflex in 6 patients. A direct relation between loss of oral sensation and control of gag reflex has been established.

Adolescent↗

The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D.

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.

Amino Acid Sequence↗

Phylogenetic analysis of gag genes from 70 international HIV-1 isolates provides evidence for multiple genotypes.

OBJECTIVE: To determine the extent of genetic variation among internationally collected HIV-1 isolates, to analyse phylogenetic relationships and the geographic distribution of different variants. DESIGN: Phylogenetic comparison of 70 HIV-1 isolates collected in 15 countries on four continents. METHODS: To sequence the complete gag genome of HIV-1 isolates, build multiple sequence alignments and construct phylogenetic trees using distance matrix methods and maximum parsimony algorithms. RESULTS: Phylogenetic tree analysis identified seven distinct genotypes. The seven genotypes were evident by both distance matrix methods and maximum parsimony analysis, and were strongly supported by bootstrap resampling of the data. The intra-genotypic gag distances averaged 7%, whereas the inter-genotypic distances averaged 14%. The geographic distribution of variants was complex. Some genotypes have apparently migrated to several continents and many areas harbor a mixture of genotypes. Related variants may cluster in certain areas, particularly isolates from a single city collected over a short time. CONCLUSIONS: The genetic variation among HIV-1 isolates is more extensive than previously appreciated. At least seven distinct HIV-1 genotypes can be identified. Diversification, migration and establishment of local, temporal 'blooms' of particular variants may all occur concomitantly.

Africa↗

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG in human serum using recombinant gag-env hybrid protein as antigen.

A novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG (anti-HTLV-1 IgG) in human serum using recombinant gag(14-139)-env-(197-295) hybrid protein is described. Anti-HTLV-1 IgG in test serum was reacted with dinitrophenyl biotinyl bovine serum albumin-recombinant gag-env hybrid protein conjugate. The complex formed was trapped onto polystyrene balls coated with affinity-purified antidinitrophenyl group IgG. After washing to eliminate nonspecific IgG in the test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with streptavidin. After washing, anti-HTLV-1 IgG in the complex trapped onto the streptavidin-coated polystyrene balls was reacted with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. By transfer of the complex, the nonspecific binding of nonspecific human IgG was considerably reduced, and the detection limit of anti-HTLV-1 IgG in serum was lowered 30-300-fold compared with that by Western blotting, gelatin particle agglutination, and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with test serum and, after washing, with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Usefulness of the immune-complex-transfer enzyme immunoassay was demonstrated using 271 serum samples.

Blotting, Western↗

Methyl-glyoxal bis guanyl hydrazone (methyl-GAG, MGBG) in advanced breast cancer. A Phase II trial of the Southwest Oncology Group.

The Southwest Oncology Group has evaluated methyl-GAG on a weekly schedule among patients with metastatic breast cancer. Among 72 fully and partial evaluable patients, one complete and four partial responses were seen. Toxicity was similar to other trials with this compound except for thrombocytopenia which was more frequent and severe and probably related to tumor infiltrating marrow. In addition, one patient experienced recall dermatitis following methyl-GAG. This toxicity has not been previously reported with this compound. Methyl-GAG has minimal activity at this dose and schedule among heavily pretreated patients with breast cancer.

Adult↗