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Biomedical subjects

H W Sheppard

Publications and source records attributed to H W Sheppard.

At least 73 records · Page 4Linked to original sources
PubMed1990

Application of a rapid microplaque assay for determination of human immunodeficiency virus neutralizing antibody titers.

To perform a human immunodeficiency virus (HIV) plaque assay in nonadherent host cells, we developed a novel technique in which HIV-infected MT-2 cells were formed into monolayers by centrifugation through molten agarose. Infection, formation of cell monolayers, and enumeration of plaques all took place in 96-well microtiter plates. When this process was preceded by 18 h of incubation of HIV with patient serum samples, neutralizing antibody titers between 1:10 and 1:5,000 could be accurately determined in patient serum samples. In addition to the determination of neutralizing antibody titers (with the use of various serum dilutions and a constant virus concentration), neutralization indices could also be determined with different virus dilutions and a single dilution of patient serum.

Cell Line↗
PubMed1990

AIDS as immune system activation. II. The panergic imnesia hypothesis.

A hypothesis is presented in which HIV infection leads to immunodeficiency through indirect subversion of critical T cell regulatory mechanisms. Acting at the T cell receptor complex (TCR), viral components (gp120) mimic the natural ligands of CD4, molecules of the major histocompatibility complex (MHC), and deliver physiologically active, inappropriate signals resulting in generalized, uncontrolled lymphocyte activation, or "panergy." Clinical manifestations of panergy include autoimmune phenomena, lymphadenopathy, hyperglobulinemia, and symptoms mediated by lymphokines. Immunologic unresponsiveness occurs early in HIV infection prior to T cell depletion because activated cells do not respond to further stimulation. Ultimately, activation disrupts T cell homeostasis by interference with the generation of memory cells ("imnesia") and leads to net T cell loss, clonal deletion, and the development of AIDS. The clinical and immunologic features of HIV disease and AIDS are reviewed from this perspective. This hypothesis is consistent with the paucity of infected T cells, the clinical findings of both AIDS-related complex (ARC) and frank AIDS, the prolonged "incubation period," and a role for antigen-specific cofactors. Based on this view of HIV pathophysiology, therapeutic modalities should avoid immune stimulation and seek to block aberrant gp120 signals at CD4 and eliminate HIV-infected cells.

Acquired Immunodeficiency Syndrome↗
PubMed1988

AIDS as immune system activation: a model for pathogenesis.

Epidemiological evidence has implicated the human immunodeficiency virus (HIV) as the etiological agent of the acquired immunodeficiency syndrome (AIDS) (Barré-Sinoussi et al., 1983; Sarngadharan et al., 1984; Kitchen et al., 1984; Levy et al., 1984). Primary infection with HIV is accompanied by an acute flu-like illness followed by a relatively long period of asymptomatic infection, the delayed appearance of lymphadenopathy and a progressive decline in immune responsiveness. Eventually, significant reduction in T4 cell number occurs along with susceptibility to a variety of opportunistic infections. It is generally accepted that this lymphotropic retrovirus causes the depletion of the helper class of T lymphocytes (T4) by direct cytopathic effects and/or the induction of autoimmune killing of T cells (Fauci, 1987). Few objections have been raised to this model despite abundant experimental evidence that viraemia and the number of infected T cells are limited and constant throughout disease progression (Levy et al., 1985; Shaw et al., 1984; Harper et al., 1986; Duesberg, 1987). We believe that the current model for HIV-induced pathology is unsatisfactory. A hypothesis is advanced in which progressive immunodeficiency is caused by indirect mechanisms without widespread T cell infection, direct virus-mediated cytopathic effects, or autoimmunity. We propose that the latency of AIDS is not due to delayed viral expression and growth but rather to the accumulation of insults to an immune system with abnormal regulatory mechanisms induced by HIV infection of macrophages.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis.

In this report we describe the construction and analysis of a genomic library of Leishmania donovani gene segments in the bacteriophage vector lambda gt11. This cloning vector permits the expression of parasite polypeptides as fusion products with Escherichia coli beta-galactosidase. A group of 90 clones which express L. donovani antigens have been isolated from this library using various polyvalent antisera. Many of these clones appear to encode parasite membrane antigens some of which are recognized by sera from patients with visceral leishmaniasis (kala azar). Some clones reacted with specific subsets of kala azar sera while others reacted with all kala azar sera tested. In addition, some of the clones appear to encode conserved antigens that are recognized by sera from mice immunized with L. major. Preliminary characterization of five clones indicated that each one contains a distinct genetic element and that at least four encode different fusion peptides.

Animals↗
PubMed1983

Recognition of Leishmania donovani antigens by murine T lymphocyte lines and clones. Species cross-reactivity, functional correlates of cell-mediated immunity, and antigen characterization.

Studies in man and experimental animals suggest that cell-mediated immunity is of primary importance in limiting the pathogenesis of cutaneous and visceral leishmaniasis. In an attempt to determine, more directly, the role of T lymphocytes and the nature of the antigens that activate them, we have propagated antigen-specific murine T lymphocyte lines and clones that proliferate in response to antigens present on the membrane of intact Leishmania donovani promastigotes. One such line cross-reacts with membrane antigens on seven other Leishmania species and, to a lesser extent, with antigens on African procyclic trypanosomes. T lymphocyte clones that also exhibited a broad range of species cross-reactivity were isolated. About 40% of these clones had highly restricted specificity, whereas 60% were more extensively cross-reactive. The parent line and some clones passively transferred footpad DTH when injected locally, and some secreted a lymphokine activity that elicited intracellular killing of amastigotes within infected macrophages. Although the proliferative response of most clones was H-2 restricted, two clones appeared to be reactive in the presence of allogeneic antigen presenting cells. The majority of the clones appeared to recognize carbohydrate containing antigens, and absorption with solid substrate-bound lectins indicated that these antigens contained both mannose and galactose ligands. The antigenic activity was also absorbed using either of two extensively cross-reactive anti-parasite monoclonal antibodies.

Animals↗
PubMed1982

Rat kappa-chain J-segment genes: two recent gene duplications separate rat and mouse.

We have cloned DNA segments containing the Jk genes from LOUVAIN rat liver, and have determined their nucleotide sequence. Seven readily identifiable Jk-coding regions (six expressible) are evident in the rat, compared with five in the mouse (four expressible). The two additional J segments in the rat appear to be the result of two sequential gene duplications occurring since the divergence of rats and mice. The first involved a homologous but unequal crossing-over in a 14 bp region spanning the 3' end of the coding region of J1 and J2. The second involved a crossing-over following unequal pairing of the two newly duplicated regions. We propose that the probability of a second duplication was greatly increased following the first as a result of the increased target for unequal pairing (370 bp of good homology versus 27 bp in the original pairing). Comparisons of rat and mouse J genes show a surprisingly high degree of sequence conservation, both inside and outside the coding regions, similar to the pattern we reported previously for the kappa constant-region gene. This provides additional evidence that constraints exist on the nucleotide sequences of these genes independent of the function of the encoded proteins.

Animals↗
PubMed1981

Allelic forms of rat kappa chain genes: evidence for strong selection at the level of nucleotide sequence.

The genes that code for two allotypic forms of the rat kappa light chain constant region (C(kappa)) have been cloned and the nucleotide sequence of 1172 base pairs of coding and flanking sequence has been determined for both alleles. These sequences have been compared to each other and to the corresponding sequences found in the mouse and human. Comparison of the LEW allele with mouse C(kappa) reveals two surprising features: (i) There is an unusually large number of amino acid substitutions (21) relative to the total number of nucleotide changes (37) in the coding region. Comparision among several other mammalian genes reveals a larger proportion of "silent" changes. (ii) The rate of accumulation of base substitutions is the same within the coding region as it is in some 870 base pairs of noncoding sequence (including 3' untranslated, 3' flanking, and 5' intervening sequences). Comparison of the two allelic forms of rat C(kappa) shows the same unusual features in more extreme form. (i) Twelve base substitutions in the coding region determine 11 amino acid differences-only one "silent" change exists. (ii) There are 12 base substitutions in the 318 base pairs of coding sequence (3.7% difference) and only 9 in the remaining 854 base pairs of noncoding DNA (1.1%), a highly significant difference. This degree of conservation of noncoding sequences and of "silent" sites within the coding region is unique among the mammalian genes studied thus far. These patterns suggest that there has been strong selection for conservation of nucleotide sequences, both inside and outside the coding region, independent of the selection required to maintain the function and characteristic structure of the immunoglobulin domain itself. The functions of the nucleotide sequences that account for this selective pressure are unclear at the present time.

Alleles↗
PubMed1977

Effects of alpha-fetoprotein on murine immune responses. II. Studies on rats.

In vitro and in vivo experiments failed to identify a consistent "immunosuppressive" or "immunoregulatory" role for alpha-fetoprotein (AFP) in the rat. Both normal and AFP-rich sera inhibited Con A, PHA, and MLR proliferation responses of lymphocyte cultures in vitro equally well, in spite of up to 100,000-fold differences in AFP concentration. AFP-rich sera also had no effect on the antibody response of the rat in vivo. Purified AFP from amniotic fluid inhibited the PHA response at concentrations of 100 in chemical contexts is converted to microgram/ml. Purified albumin was also inhibitory, but at 1 log higher concentration. Purified AFP from tumor sera was not inhibitory at 100 in chemical contexts is converted to microgram/ml for Con A, PHA, or MLR reactions and only inhibited PHA stimulation at medium concentrations greater than 1000 in chemical contexts is converted to microgram/ml. The role of AFP as an immunosuppressive agent, particularly in reference to survival of mammalian fetuses, should be reconsidered.

Animals↗
PubMed1976

In vitro studies of the rabbit immune system. V. Suppressor T cells activated by concanavalin A block the proliferation, not the induction of antierythrocyte plaque-forming cells.

The late B-cell proliferative phase of the in vitro antibody response by rabbit spleen cells is highly susceptible to suppression by activated T cells. The in vitro antisheep erythrocyte plaque-forming cell (PFC) response by spleen cells from normal or primed rabbits can be suppressed by adding concanavalin A (Con A), Con A-prestimulated peripheral blood or spleen lymphocytes, or supernates from Con A-prestimulated peripheral blood lymphocytes. The suppression is not mediated by a direct interaction of Con A with responding cells as shown by the effectiveness of prestimulated cells. Primed spleen cultures remain sensitive to Con A suppression as late as 72 h after initiation, and the addition of Con A after 24-72 h rapidly stops the increase in the number of PFC. T cells are required for Con A addition to be effective but the suppression can be induced at a time when T-helper cells are no longer necessary. Further, the suppressive effect of Con A addition is abrogated by specific antisera to rabbit T cells. We propose that Con A activates suppressor T cells which then exert their effects on proliferating PFC or their immediate precursor B cells. The early inductive or recruitment phase of the response is probably not blocked by suppressor cells. Also, there is an apparent relationship between the number of proliferating B cells and the number of suppressor cells required. Finally, the difficulties in inducing a stimulatory effect by Con A and the prolonged period that Con A addition is suppressive suggests that the rabbit has relatively more and/or longer-lived suppressor cells than the mouse and may be a particularly useful species for studying suppressive phenomena and their mechanisms.

Animals↗
PubMed1976

Effect of anti-alpha1-fetoprotein on alpha1-fetoprotein-producing rat tumors in vivo and in vitro.

Active or passive immunization of rats to alpha1-fetoprotein (AFP) does not consistently inhibit the growth of AFP-producing transplantable hepatomas in vivo, and anti-AFP does not kill these hepatomas in vitro. However, 3 of 14 rats in 1 experiment responded to passive immunization by reversal of tumor growth as evidenced by normalization of elevated AFP serum concentrations, and 1 of 9 rats actively immunized with rat AFP in complete Freund's adjuvant had suppressed growth of transplantable hepatoma 7777.

Animals↗