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J Guyden

Publications and source records attributed to J Guyden.

14 recordsLinked to original sources
PubMed2001

Questionable thymic nurse cell.

Since their discovery in 1980, thymic nurse cells (TNCs) have been controversial. Questions pertaining to the existence of the TNC as a "unit" cell with thymocytes completely enclosed within its cytoplasm were the focus of initial debates. Early skeptics proposed the multicellular complex to be an artifact of the procedures used to isolate TNCs from the thymus. Since that time, TNCs have been found in fish, frogs, tadpoles, chickens, sheep, pigs, rats, mice, and humans. Their evolutionary conservation throughout the animal kingdom relieved most speculations about the existence of TNCs and at the same time demonstrated their apparent importance to the thymus and T-cell development. In this review we will discuss and debate reports that describe (i) the organization or structure of TNCs, (ii) the thymocyte subset(s) found within the cytoplasm of TNCs and their uptake and release, and (iii) the function of this fascinating multicellular interaction that occurs during the process of T-cell development. Discussions about the future of the field and experimental approaches that will lead to answers to remaining questions are also presented.

Animals↗
PubMed2000

A single point mutation in HIV-1 V3 loop alters the immunogenic properties of rgp120.

The results of the study presented in this report show that clones of env derived from genetically divergent HIV-1 field isolates fall into two major subsets based on the predicted secondary structure of the V3 region in gp120. One subset exemplified by the clones A-UG06c, B-RT3.12 and C-UG045 is predicted to assume a beta-turn conformation in the V3 loop and comprises the GPGX residues. The other subset exemplified by the clones D-UG23c and D-UG042 (GXGX) are deficient in the expression of the beta-turn in the loop. Since secondary conformations are highly likely to confer antigenic properties in a protein backbone at least for B cells, we have used nucleic acid immunization to test the effect of the beta-turn deficiency on the immunogenic potential of rgp120 encoded in these field isolates. As hypothesized, inoculation of BALB/c mice with the env plasmid encoding the beta-turn expressing rgp120 molecules resulted in the development of a vigorous antibody response to the homologous V3 loop peptides. In contrast, immunization with an rgp120 clone deficient in the beta-turn in the V3 loop showed no evidence of antibody development to the V3 loop. Instead, the latter clones triggered T cell proliferative responses and markedly increased the level of IL-2 and IFN-gamma production by T cells. Significantly, reconstitution of the beta-turn conformation by site-directed mutagenesis of a single V3 loop residue yielded rgp120 molecules which restored antibody production while diminishing the cell-mediated immune (CMI) responses to the V3 residue. These observations demonstrate the marked impact of a single amino acid substitution on the immunogenic properties of V3 region in gp120 encoded by divergent HIV-1 field isolates.

Amino Acid Sequence↗
PubMed1996

Immunogenic potential of rgp120 from African HIV-1 subtype A.

Previous studies have shown that the African strains of HIV-1 mostly cluster with the subtypes A, C or D based on phylogenetic analysis of the ENV nucleotide sequences. In the present investigation we have examined the immunogenic potential of full length gp120 derived from the Ugandan HIV-1 subtype A isolate, AUG06c, using computer-based prediction methods and a plasmid-mediated immunization technique. Computer-assisted analysis of the amino acid residues identified 15 potential B-cell epitopes in gp120 of AUG06c. Despite marked variation in the primary sequences, these epitopes were shown to correspond well to analogous sites in gp120 derived from the subtype B reference clones, MN and IIIBBH10. The relative positions of the epitopes indicated that E9[V3], E14[C3] and E15[V5] correspond to the previously defined principal neutralizing determinant (PND) located in the V3 loop, the CD4 binding site and gp120 "immunodominant" region, respectively. Intramuscular inoculation of BALB/c mice with the ENV clones from AUG06c or from the subtype C clone, CUG045 elicited antibodies which react with the homologous but not with the heterologous PND peptide in ELISA. However, cocktail inoculation with the ENV plasmids from AUG06c and CUG045 elicited antibodies which reacted with both peptides. Antibody response to the other predicted epitopes of AUG06c was not as strong as the response to the PND peptide. The response of the mice to DNA-mediated immunization was further tested in a proliferation assay. Spleen cells derived from the immunized mice exhibited a strong proliferative response to homologous and heterologous PND peptides in [3H]thymidine incorporation assay. DNA-mediated immunization with rgp120 of AUG06c appears to elicit cellular immune response of relatively broad specificity.

Amino Acid Sequence↗
PubMed1995

Relative reactivity of the V3 loop PND of HIV-1 subtypes A, B, C, D, and F with sera from selected Ugandan localities.

Synthetic peptides comprising the predicted principal neutralizing determinant (PND) in new African and North American HIV-1 clones were tested in ELISA for reactivity with ninety six serum samples from asymptomatic donors in six selected localities in Uganda. Irrespective of the geographical origin of the samples, the majority of the test sera cross-reacted at high intensities with the peptides derived from the North American clone, BRT3.6 (Group B), the Ugandan clone, CUG045, (Group C), and the Romanian clone, FRMA (Group F). The frequency of reactivity of the peptides from BRT3.6, CUG045, and FRMA were within the ranges of 57-100%, 50-100%, and 57-100%, respectively, for the sera collected from these disparate localities. In contrast to these findings, the V3 peptides derived from the other Ugandan isolates showed a more restricted pattern of reactivity with the same serum samples: AUG06c (1-63%), DUG23c (2%), and DUG044 (38-87%). The results from ELISA inhibition assay indicated that the V3 peptide from BRT 3.6, CUG045, and FRMA express closely related antigenic specificities quite distinct from those in AUG06c and DUG044. The residues comprising the PND in BRT 3.6, CUG045, and FRMA appear to be well conserved in the HIV-1 subtypes prevalent in the selected Ugandan locales.

Amino Acid Sequence↗
PubMed1995

Distribution of linear antigenic epitopes on GP120 encoded in sibling clones of novel New York HIV-1 subtype B isolates.

We have initiated studies to characterize the predominant subtypes of HIV-1 which account for infections in a defined cohort of intravenous (IV) drug addicts. A region of ENV encoding the C2 to the V5 regions was amplified from the leukocytes of two subjects currently enrolled in a methadone maintenance program at the Addiction Research and Treatment Corporation (ARTC), in Brooklyn, New York. This region of the viral genome encodes the principal neutralizing determinant (PND) located in the V3 loop, the immunogenic CD4-binding site, and six other linear antigenic epitopes in the envelope glycoprotein, gp120. Phylogenetic tree analysis of the nucleotide sequences showed that the sibling clones RT1.4, RT1.15, RT1.17, RT1.21 and RT3.6, RT3.10, RT3.11, RT3.12 and RT3.15 derived from the isolates, RT1 and RT3, respectively, cluster with "group B" viruses at 99% confidence level. Marked intra-patient and inter-patient sequence variation was apparent in the V3 loop. The divergence included the presence of a previously unreported hexapeptide GPWGTF at the cap of the loop in the clones from RT1. The North American consensus hexapeptide, GPGRAF, was identified in the cap of the loop from the clones of RT3. Four of the five sibling clones from RT3 were closely related whereas the other clone, RT3.15, displayed five amino acid mutations downstream of the V3 cap. To assess the effect of sequence variation on the distribution of linear antigenic epitopes, complementary computer software programs, were used to analyze the gp120 residues. Eight analogous antigenic epitopes were identified in the clones from both isolates despite the marked divergence in the primary sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The binding, internalization, and release of thymocytes by thymic nurse cells.

Recent studies in our laboratory have described the development of the SV40-transformed thymic nurse cell (TNC) line SVT-II2, that maintains the ability to internalize thymocytes in vitro. SVT-II2 cells were shown to bind and internalize a subset of the alpha beta TCR+, CD4+CD8+ thymocyte population exclusively. Also, SVT-II2 cells express cell surface class I and class II MHC antigens. These data are consistent with reports that suggest that TNCs may have a role in thymic education. In this report, we used scanning electron microscopy, transmission electron microscopy, and long-term video microscopy to study binding, internalization, and release of thymocytes by TNCs. The results of these experiments showed the internalization event to be selective and dynamic. The process appears to involve programmed cooperation between the two cell types that terminates with the release of selected thymocytes. Although no changes in thymocyte cell surface phenotype were detected as a result of their interaction with TNCs in vitro, over 90% remained viable after a 48-hr incubation period.

Animals↗
PubMed1993

Independent divergences in the CD4 binding site and V3 loop encoded in two seroprevalent Ugandan HIV-1 clinical isolates.

Two major epitopes expressed in HIV-1 have been recently shown to play a central role in virus neutralization. One of these important specificities is a type-specific or group-specific, principal neutralizing determinant (PND) located in the V3 loop of gp120. The other is a more broadly neutralizing determinant associated with the CD4 binding site. Structural and serological studies of the variation in these epitopes have become important in vaccine research. This report describes the analysis of the DNA clones encoding a region of gp120 that overlaps the V3 loop and the putative CD4 recognition site in two new African isolates, UG06c and UG23c. Phylogenetic analyses of the DNA sequences showed that the new African isolates clustered with two very distinct subtypes of HIV-1. UG06c was grouped with U455, D687, and Z321, previously classified as "HIV-1 subtype A" in the AIDS and human retroviruses database; and UG23c was grouped with MAL, JY1, NDK, ELI, and Z2Z6 classified as "HIV-1 subtype D." Considerable variation was apparent in the V3 loop. The divergence included the presence of the hexapeptides GP-GRSF and GLGQAL at the cap of the loop in UG06c and UG23c, respectively. The GPGR tetrapeptide in UG06c formed a beta-turn configuration similar to that of MN or IIIB. The beta-turn was not found to be a likely conformation for GLGQ. The amino acids previously implicated in CD4 binding and the associated neutralizing activity were relatively conserved. To assess a possible impact of the sequence and conformational variations on serological reactivity, UG06c and UG23c were subjected to neutralization assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.

Animals↗
PubMed1991

The immortalization of thymic nurse cells by SV40 virus.

Thymic nurse cells (TNCs) are stromal elements that contain between 20 and 200 T cells within their cytoplasm. Because of this unique feature they are believed to play a role in thymocyte development. Unfortunately, it has been difficult to obtain pure TNCs in quantities sufficient for extensive evaluation of their thymic function. As a result, only a limited amount of information is available that characterizes TNCs or the T cell population(s) found within their cytoplasm. We have now used SV40 to infect and immortalize TNCs from C57BL/6 mice. SV40-transformed TNCs were found to specifically bind and internalize cells from an immature thymocyte line isolated in our laboratory. These results describe a method of obtaining pure populations of TNCs for future studies of their thymic function, and suggest that binding to specific subpopulations of lymphoblasts may be necessary for internalization.

Animals↗
PubMed1989

Neutral selection of transfected mammalian cells using tissue plasminogen activator gene expression.

The gene that produces the proteolytic enzyme, tissue plasminogen activator (tPA), was used as a selectable marker for transfection. Cells that were successfully transfected with plasmids containing a gene for tPA (tpa) produce plaques in the caseinolysis assay. Two tpa-containing plasmids were constructed and used to transfect a variety of cell types. One plasmid contained the promoter region of simian virus 40 and the other contained the Moloney murine leukemia virus promoter. No significant difference in transfection frequencies was found for the two plasmids. However, 80% of the cell types tested produced plaques. This system allows for the identification and isolation of transfected cells under conditions that are not lethal to nontransfected cells.

Animals↗
PubMed1984

Immunospecific vesicle targeting facilitates fusion with selected cell populations.

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts or liposomes, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte and monocyte cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. Attachment of vesicles to selected cells was achieved by first labelling the cells with biotin-modified antibody and then treating them with avidin-coupled sealed ghosts or liposomes. Another approach to the promotion of selective fusion aims to alter the cell recognition properties of Sendai virus so that its fusogenic activity may be redirected to specific cellular targets. The agglutination and fusion of red cells by UV-inactivated Sendai virus were completely blocked by low concentrations of a Fab preparation of a monoclonal antibody against the viral haemagglutinin (HN) sites. Agglutination and fusion activity were restored in the presence of Fab-anti-HN by providing an alternative recognition system, namely, when the virus had been coupled with biotin and the red cells with avidin. Methods for facilitating microinjection by specifically directing vesicles to target cells may be particularly useful in overcoming barriers to the transfer of genes into lymphocytes by standard transfection techniques.

Animals↗
PubMed1980

A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation.

Cells infected by one strain of Fujinami sarcoma virus (FSV) are transformed at 38 degrees C but are phenotypically normal at 41.5 degrees C. FSV encodes a 140,000 molecular weight protein (P140) with gag gene-related and FSV-specific peptide sequences. At 41.5 degrees C, P140 is weakly phosphorylated at serine residues, and is inactive in the immune complex protein kinase assay. At 38 degrees C, P140 is highly phosphorylated, contains phosphotyrosine in addition to phosphoserine, and in the immune complex kinase assay becomes phosphorylated at three tyrosine residues. Phosphorylation of cellular polypeptides at tyrosine residues in FSV-infected cells is also temperature-sensitive. These observations indicate that P140 is the transforming protein of FSV and that protein phosphorylation at tyrosine residues is involved in transformation by this virus.

Alpharetrovirus↗
PubMed1978

Metabolism of benzo(a)pyrene in animals with high aryl hydrocarbon hydroxylase levels and high rates of spontaneous cancer.

Ambystoma tigrinum found in a sewage polluted pond had high levels of aryl hydrocarbon hydroxylase (AHH) activity that decreased to the basal level of control animals after being held several months in clean water. The qualitative formation of benzo(a)pyrene (BP) metabolites by salamander hepatic microsomes was similar to those seen for other species. Inhibition of epoxide hydrase activity did not alter the total metabolite production but did change the ratio of metabolites. A correlation appears to exist between high AHH induction, the presence of polycyclic hydrocarbon pollutants, and the high rate of spontaneous cancer in salamanders.

Ambystoma↗