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Biomedical subjects

M A Derby

Publications and source records attributed to M A Derby.

11 recordsLinked to original sources
PubMed1999

Interaction of murine MHC class I molecules with tapasin and TAP enhances peptide loading and involves the heavy chain alpha3 domain.

In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections.

We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). 1) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T-lymphocyte (CTL) epitopes was found to be important. 3) We developed an "epitope enhancement" approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules. 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High-avidity CTLs, however, were found to undergo apoptosis when confronted with high-density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF-RII, and a permissive state induced through the T-cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines. 6) Intrarectal immunization with peptide vaccine induced mucosal and systemic CTL. Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin-12. 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre-existing vaccinia immunity for use of recombinant vaccinia vaccines.

AIDS Vaccines↗
PubMed1998

Supraoptimal peptide-major histocompatibility complex causes a decrease in bc1-2 levels and allows tumor necrosis factor alpha receptor II-mediated apoptosis of cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) are primary mediators of viral clearance, but high viral burden can result in deletion of antigen-specific CTLs. We previously reported a potential mechanism for this deletion: tumor necrosis factor (TNF)-alpha-mediated apoptosis resulting from stimulation with supraoptimal peptide-major histocompatibility complex. Here, we show that although death is mediated by TNF-alpha and its receptor (TNF-RII), surprisingly neither the antigen dose dependence of TNF-alpha production nor that of TNF-RII expression can account for the dose dependence of apoptosis. Rather, a previously unrecognized effect of supraoptimal antigen in markedly decreasing levels of the antiapoptotic protein Bc1-2 was discovered and is likely to account for the gain in susceptibility or competence to sustain the death signal through TNF-RII. This decrease requires a signal through the TCR, not just through TNF-RII. Although death mediated by TNF-RII is not as widely studied as that mediated by TNF-RI, we show here that it is also dependent on proteolytic cleavage by caspases and triggered by a brief initial encounter with antigen. These results suggest that determinant density can regulate the immune response by altering the sensitivity of CTLs to the apoptotic effects of TNF-alpha by decreasing Bc1-2 levels.

Animals↗
PubMed1998

Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge.

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer's patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein of Mr 160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-gamma dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.

AIDS Vaccines↗
PubMed1983

Identification of sub-populations of chick neural retinal cells by monoclonal antibodies: a fluorescence activated cell sorter screening technique.

An assay was developed which identifies monoclonal antibodies recognizing the cell surfaces of sub-populations of chick retinal cells which survive in culture. Antibodies from hybridoma culture supernatants were bound to monolayers of retina cells followed by a fluorescent secondary antibody. The quantitative fluorescence analysis ability of the fluorescence-activated cell sorter (FACS) was used to determine the fluorescence intensity associated with viable single cells and the frequency with which these cells appear in the total population. Hybridomas were generated which define both overlapping and non-overlapping retina cell populations. The cytotoxic activity of many of the monoclonal antibodies was determined on the FACS by propidium iodide exclusion, and the identity of various sub-populations was demonstrated by immunohistochemical staining of retina sections.

Animals↗
PubMed1983

Monoclonal antibodies against a differentiated retinal cell population.

Two monoclonal antibodies have been isolated which bind preferentially to the plexiform layers of embryonic chick neural retina and to 50-60% of dissociated neural retinal cells in culture as determined by surface binding to cells followed by analysis in a fluorescent activated cell shorter. Each antibody appears to recognize a distinct antigenic determinant on a common cell surface antigen, a protein of approximately 230 kdalton. This antigen increases dramatically in concentration between embryonic days 7 and 11 concomitant with the elaboration of the retinal plexiform layers. The antigen appears first in the central portion of the neural retina and at later times in the periphery, an appearance consistent with the normal pattern of differentiation of the retina.

Animals↗
PubMed1982

Differentiation of avian neural crest cells in vitro: absence of a developmental bias toward melanogenesis.

Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (greater than 400K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.

Animals↗
PubMed1982

Environmental factors affecting neural crest differentiation: melanocyte differentiation by crest cells exposed to cell-free (deoxycholate-extracted) dermal mesenchyme matrix.

Deoxycholate-extracted, cell-free matrices were prepared from primary explants or dispersed cell cultures of embryonic avian dermis, ectoderm, gut mesenchyme, endoderm, pharynx, or umbilical artery. Neural crest cells in association with matrices from dermal explants or monolayers formed melanocytes after six days. Crest cells in association with matrices from all other tissues or grown on plastic did not form melanocytes. It is concluded that a deoxycholate-resistant structural component of the dermal extracellular matrix induces melanocyte differentiation.

Animals↗
PubMed1978

The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC.

Treatment of tissue sections with enzymes wich degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. Both Streptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of both Streptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, and Streptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.

Alcian Blue↗