Biomedical subjects

J Hackett

Publications and source records attributed to J Hackett.

At least 73 records · Page 4Linked to original sources

PubMed1989

Identification of a subset of murine natural killer cells that mediates rejection of Hh-1d but not Hh-1b bone marrow grafts.

NK cells demonstrate many immune functions both in vitro and in vivo, including the lysis of tumor or virus-infected cells and the rejection of bone marrow allografts. However it remains unclear whether or not all NK cells can mediate these various functions or if NK cells exist in functionally distinct subsets. We have developed a new NK-specific mAb, SW5E6, which binds to approximately 50% of murine NK cells. The 5E6 antigen identifies a distinct and stable subset of NK cells and is expressed on about one-half of fresh or rIL-2-activated murine NK cells. Both 5E6+ and 5E6- NK cells are capable of lysing YAC-1 tumor cells in vitro and in vivo. By treating animals with SW5E6, we demonstrate that the 5E6+ subset is necessary for the rejection of H-2d/Hh-1d but not H-2b/Hh-1b bone marrow cells. Thus NK cells exist as functionally separable subsets in vivo.

Animals↗

PubMed1989

Inhibition of immunoglobulin gene rearrangement by the expression of a lambda 2 transgene.

The rearrangement of Ig genes is known to be regulated by the production of H and kappa L chains. To determine whether lambda L chains have a similar effect, transgenic mice were produced with a lambda 2 gene. It was necessary to include the H chain enhancer, since a lambda gene without the added enhancer did not result in transgene expression. The lambda 2 transgene with the H enhancer was expressed in lymphoid cells only. The majority of the B cells of newborn transgenic mice produced lambda, whereas kappa + cells were reduced. Concomitantly, serum levels of kappa and kappa mRNA were diminished. By 2 wk after birth the proportion of kappa-expressing cells was dramatically increased. Adults had reduced proportions of B cells that produced lambda only, but the levels of lambda were still higher than in normal littermates. Also, kappa + cells were still lower than in normal mice. Analysis of hybridomas revealed that reduction of kappa gene rearrangement was the basis for the decreased frequency of kappa + cells. Furthermore, many cells also contained an unrearranged H chain allele. It was concluded that feedback inhibition by the lambda 2 together with endogenous H protein may have inhibited recombinase activity in early pre-B cells, leading to inhibition of both H chain and kappa gene rearrangement. Thus, lambda 2 can replace kappa in a feedback complex. The levels of serum lambda 1 and, to a lesser degree, of spleen lambda 1 mRNA were reduced in the lambda 2 transgenic mice. However, the proportion of hybridomas with endogenous lambda gene rearrangement was at least as high as in normal mice. It was therefore concluded that the suppression of functional lambda 1 may be a consequence of decreased selection of endogenous lambda-producing cells because of the excess of transgenic lambda. The escape of kappa-producing cells from feedback inhibition may be the result of several mechanisms that operate to varying degrees, among them: (a) kappa rearrangement during a period in which the recombinase is still active after appearance of a lambda 2/mu stop signal; (b) a B cell lineage that is not feedback inhibited at the pre-B cell stage; (c) subthreshold levels of transgenic lambda 2 in some pre-B cells; and (d) loss of the lambda 2 transgenes in rare pre-B cells.

Animals↗

PubMed1989

Natural killer cells and their precursors in mice with severe combined immunodeficiency.

Our studies with scid mice have clarified the relationship between T cells and NK cells. C.B-17 scid mice have normal frequency of transplantable NK progenitors in their bone marrow which develop into fully functional NK cells. Spleens of scid mice contain mature NK cells which are phenotypically and functionally indistinguishable from NK cells found in normal mice. These cells retain their TCR genes in germline configuration and do not transcribe the CD3 genes. Thus, NK cells are distinct from the earliest identifiable cells committed to the T-lineage. In addition to the spleen, the thymus of scid mice also contains mature NK cells. These cells constitute a small proportion of the thymus cell population and can be clearly distinguished from the majority of cells, which have the phenotype and molecular characteristics of very early T-lineage cells. There is no evidence that NK cells within the thymus are derived in situ from a common NK/T precursor. Together these data support the hypothesis that NK cells form an independent lineage.

Animals↗

PubMed1989

Molecular cloning and analysis of the incompatibility and partition functions of the virulence plasmid of Salmonella typhimurium.

The incompatibility functions (inc) of the virulence plasmid of Salmonella typhimurium LT2 were initially located in a 4.3 kb region near the repA locus of the plasmid. Expression of inc required the presence, in cis or in trans, of two distinct DNA regions of the fragment. These regions, maximally 0.3 kb and 0.6 kb in size, were separated in the fragment by c. 3.0 kb. This intervening DNA encoded two proteins, of Mr values 37 kDa and 40 kDa. The promoter for the 37 kDa protein lay in or near one of the inc regions (incL). No function was assigned to this protein, however, it may be the product of a rep gene. The 40 kDa protein may have a partition (par) function, and may bind to a centromeric site in or near the other inc region (incR). An inc+ par- derivative of the original plasmid clone was used in a simple method, not involving the use of curing agents/mutagens, to eliminate virulence plasmid DNA from Salmonella typhimurium, Salmonella dublin, Salmonella enteritidis, and Salmonella cholerae-suis. The par function served to stabilise pJRD158B-based plasmid greater than 10(6)-fold in Escherichia coli and the virulence plasmid-cured Salmonella strains.

Cloning, Molecular↗

PubMed1989

Pan natural killer cell monoclonal antibodies and their relationship to the NK1.1 antigen.

The study of natural killer (NK) has been difficult because they account for a small percentage of peripheral blood and splenic lymphocytes and the paucity of NK specific antigens that have been identified. We have isolated pure populations of C57BL/6 (H-2b) NK cells using the IgG2b monoclonal antibody PK136 (anti-NK1.1). These NK1.1+ cells were used to immunize 129/J (H-2b) mice, and in this report, we describe three new NK specific monoclonal antibodies (SW3A4(IgM), SW4B12(IgG1), and SW2B4(IgG2b] and their relationship to the known murine NK antigen NK1.1. We have further characterized the NK1.1 antigen as a 39 kd molecule which is coded for by a gene which appears to map to chromosome 6.

Animals↗

PubMed1988

Retinal insulin receptors: localization using a polyclonal anti-insulin receptor antibody.

Although retinal insulin receptors have recently been described biochemically, the location of these receptors within the retina is unknown. The study presented here used a polyclonal anti-insulin receptor antibody (B10), immunofluorescence and immunoelectron microscopy to determine the location of insulin receptors in bovine, monkey and human retina. It was found that antibody immunofluorescence formed discrete bands localized predominantly to photoreceptor and neuronal cell bodies. In addition to the strong association with neuronal perikarya, a lower level of antibody binding was observed in photoreceptor outer segments. In human retina, some of the antibody immunofluorescence also had a pattern that suggested B10 binding to glial-like cells.

Animals↗

PubMed1988

Towards a live oral vaccine against enterotoxigenic Escherichia coli of swine.

A live oral vaccine has been developed against scouring induced in piglets by enterotoxigenic Escherichia coli (ETEC). An attenuated strain of Salmonella typhimurium, G30, has been used as a vector for plasmids encoding the production of the fimbrial colonization factors of porcine ETEC. Initial studies with clones expressing K88 or K99 fimbriae have shown them to be well tolerated when administered orally in very high doses. The clones elicited serum, colostral and milk antibodies to the fimbrial antigens, and a challenge trial indicated that such responses were sufficient to ensure the passive transfer of protective immunity to suckling piglets. The possible advantages of this approach are discussed.

Administration, Oral↗

PubMed1988

A chromosomal integration system for stabilization of heterologous genes in Salmonella based vaccine strains.

We have developed a system whereby heterologous DNA encoding an antigen from an enteropathogen may be recombined into the chromosome of an attenuated Salmonella carrier strain. The system involves two steps: (i) integration of a hisOG deletion mutation into the chromosome; (ii) replacement of the hisOG deletion by the complete hisOG region and the segment of heterologous DNA which encodes the antigen of interest. Recombinants may be selected (his+). The system was used to integrate the genes encoding K88 fimbriae from enterotoxigenic Escherichia coli into the chromosome of a galE mutant of Salmonella typhimurium (LT2H1). Recombinants were detected at a frequency of between 1.0 x 10(-3) and 1.5 x 10(-3). A variety of tests confirmed that the K88 genes were integrated into the chromosome of LT2H1 and were expressed. The stability of the recombinant was tested both in vivo and in vitro. When administered orally to mice, the recombinant elicited a serum antibody response to K88, and retained the Salmonella vaccine potential of the vector strain.

Antigens, Bacterial↗

PubMed1988

Oral immunization with live, avirulent fla+ strains of Salmonella protects mice against subsequent oral challenge with Salmonella typhimurium.

Some strains of Salmonella, when fed to mice, establish a nonlethal, limited infection in the Peyer's patches of the small intestine. When such mice are later challenged orally with a normally lethal dose of virulent Salmonella typhimurium, a protective effect of the prior vaccination is seen. In an effort to characterize the determinant(s) of the avirulent strains responsible for this protective effect, we examined the cell envelope protein profiles of five such protective strains and of eight strains of Salmonella that were both nonpersistent and nonprotective when fed to mice. The protective strains produced high levels of flagellin. We made otherwise isogenic fla+ and fla- derivatives of two such strains and showed that although the fla- derivatives colonized mice as well as did the fla+ strains, the fla- derivatives given orally showed a much reduced ability to protect mice from S. typhimurium challenge. Both fla+ and fla- strains induced cellular immunity in vaccinated mice.

Administration, Oral↗

PubMed1988

Phenotypic heterogeneity and cytotoxic activity of Con A and IL-2-stimulated cultures of mouse Thy-1+ epidermal cells.

Short-term and long-term cultures of mouse Thy-1+ epidermal cells (EC) were established in order to characterize their phenotypic and functional properties. Concanavalin A (Con A) and Interleukin 2 (IL-2) stimulated Thy-1+ EC mediated non-MHC directed cytotoxicity preferentially against the NK-sensitive target, YAC-1 vs the NK-resistant target, P815; these cells also mediated antibody-dependent cell-mediated cytotoxicity (ADCC), indicating the presence of IgG-FcR on at least some of them. Freshly isolated Thy-1+ EC failed to lyse YAC-1 targets; however, this activity was observed after 9 d of culture with Con A and IL-2. While dendritic Thy-1+ EC, in vivo, do not express the T-cell markers, L3T4 and Lyt-2, short-term cultured cells displayed phenotypic heterogeneity with small but significant percentages of Lyt-2+ and L3T4+ cells appearing transiently. The phenotype of the effector cell(s), which mediates cytotoxic activity, was determined by utilizing flow cytometry to sort short-term cultured EC into positively and negatively stained populations. Cells which express L3T4, or which lack asialo GM1, did not lyse YAC-1 targets; maximum cytotoxic activity was found within populations of cells which are asialo GM1+, Lyt-2-, and asialo GM1+, Lyt-2+. These studies indicate that Thy-1+ cells derived from mouse epidermis when cultured in the presence of Con A and IL-2 have the capacity to generate a phenotypically heterogeneous population, some cells of which are capable of mediating cytotoxic activities.

Animals↗

PubMed1988

Mechanism of anti-asialo GM1 prevention of graft-vs-host disease: identification of allo-antigen activated T cells.

The purpose of this study was to begin to dissect the mechanism whereby anti-asialo GM1 (alpha ASGM1) prevents otherwise lethal graft-vs-host disease (GVHD) across multiple minor histocompatibility barriers in mice. Phenotypic characterization of cells from the peak proliferative time of the graft-vs-host reaction (C57BL/6J lymph node cells----irradiated LP/J, days 5-7) revealed the alpha ASGM1 and alpha Thy 1.2 identified cells with an approximate 80% concordance and that NK-1.1 staining was negligible. Because resting T cells do not label with alpha ASGM1, the epitope recognized by alpha ASGM1 on GVHR T cells is an activation antigen. Because asialo GM1 has been previously found on the surface of activated macrophages, we wanted to distinguish between the two most likely targets for the in vivo effect of alpha ASGM1 infusions (T cells or macrophages). We compared the effects of alpha ASGM1 infusions on alloantigen-stimulated T-cell proliferation versus antigen presentation: T-cell proliferation was markedly reduced by alpha ASGM1 infusions, whereas antigen presentation function was not diminished. We conclude that the mechanism whereby alpha ASGM1 prevents GVHD does not involve NK cells or antigen presenting cells, but does involve activated donor T cells. The potential therapeutic advantage of such an antibody for use in human disorders compared to pan-immunosuppression lies in its ability to eliminate selectively those T cells involved in the immunologic process (i.e. activated T cells) while sparing the remainder of the T-cell repertoire.

Animals↗

PubMed1988

A galE via (Vi antigen-negative) mutant of Salmonella typhi Ty2 retains virulence in humans.

We have recently described the construction of a galE derivative of Salmonella typhi Ty2 (Ty2H1) which had a 0.4-kilobase deletion in the galE gene and was sensitive to galactose-induced lysis when cultured with greater than or equal to 0.06 mM galactose (D. M. Hone, R. Morona, S. Attridge, and J. Hackett, J. Infect. Dis. 156:167-174, 1987). We now report the selection of a rifampin-resistant, via derivative of Ty2H1, EX462. Compared with the Ty2 parent strain, EX462 was serum sensitive and highly attenuated in the mouse mucin virulence assay. When four human volunteers ingested 7 X 10(8) viable EX462, two became ill and developed a typhoidlike disease with fever and bacteremia. Blood isolates from these individuals were indistinguishable from the vaccine strain by a variety of criteria. We concluded that, even in a via background, the galE mutation was not attenuating for S. typhi in humans.

Animals↗

PubMed1988

Development of the anchoring structures of the epithelium in rabbit and human fetal corneas.

The sequence of development of the components of the corneal adhesion complex (hemidesmosomes, basal lamina and anchoring fibrils) was studied in rabbit and human fetal corneas using electron microscopy and histochemical localization of type VII (anchoring fibril) collagen. In the rabbit, basal lamina was present at 15 days gestation, followed by hemidesmosomes (HDs) and anchoring fibrils (AFs) at 20 days gestation. Type VII collagen was first localized at 20 days. At 25 days, HDs remained low compared to the adult value. During human corneal development, basal lamina was present at 8 weeks gestation. Through 12 weeks of gestation, no HDs or AFs were discernible nor was there any type VII localization. At 13-19 weeks, HDs and cross-banded AFs were seen, and localization of type VII collagen was first noted. A palisade of filaments extending perpendicularly from the basal lamina into the underlying stroma was discernible from 13 to 27 weeks. A distinct Bowman's layer was present at 19 weeks. By 27 weeks, HDs/micron membrane were greater than or equal to the adult value, and AF penetration into the underlying stroma was also greater than or equal to the adult value. Bowman's layer had not reached adult values by term. These data indicate that after basal lamina deposition, HDs and AFs develop synchronously in both species. In humans the palisade of filaments may be the precursor of Bowman's layer, and the AF network develops within Bowman's layer.

Animals↗

PubMed1987

Mediation of serum resistance in Salmonella typhimurium by an 11-kilodalton polypeptide encoded by the cryptic plasmid.

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.

Animals↗

PubMed1987

Nonlipopolysaccharide protective antigens shared by classical and El Tor biotypes of Vibrio cholerae.

The prophylactic significance of the nonlipopolysaccharide (non-LPS) antigens of Vibrio cholerae was investigated further with use of the infant mouse cholera model. Of 16 strains examined to date, 12-including eight recent field isolates of both biotypes and both common serotypes-express common non-LPS protective antigens. The exceptional strains are four old isolates of El Tor biotype, the outer membrane proteins of which are not atypical when analyzed by immunoblotting. The protective activities of antibodies to the shared non-LPS components correlated with their capacities to inhibit the in vitro attachment of Vibrio organisms to isolated murine enterocytes.

Animals↗

PubMed1987

Construction of defined galE mutants of Salmonella for use as vaccines.

We describe the molecular cloning of the gal operon of Salmonella typhimurium LT2 and the localization of the gal promoter and the genes galE, galT, and galK. The order of the genes and the direction of transcription was the same as in Escherichia coli K12. A restriction enzyme map of the operon was obtained, and the approximate termini of the gal genes were located by using transposon insertion mutagenesis and minicell analysis. We constructed a plasmid that contained a defined 0.4-kilobase deletion in the galE but still expressed galT and galK activities from the gal promoter. This galE deletion was recombined into the chromosomal gal operons of S. typhimurium and Salmonella typhi Ty2. The resulting strains were vigorous, nonreverting galE mutants that were sensitive to galactose-induced lysis at 0.2 mM galactose. The S. typhimurium galE derivatives were avirulent and protective in mice.

Animals↗

PubMed1987

Immunoelectron microscopic localization of photoreceptor-specific markers in the monkey retina.

Antibodies for several molecules that function in the visual process were used to localize these molecules in primate rod and cone cells. These antibodies (monoclonal or polyclonal) were prepared against Interphotoreceptor Retinoid-binding Protein (IRBP), S-antigen (S-Ag), opsin, alpha-transducin and also against cyclic GMP (cGMP). Lowicryl-embedded tissues were labeled with secondary antibodies linked to colloidal gold. Although IRBP is predominantly an extracellular protein, the relatively small amount found intracellularly was localized mainly in rods, with little in cones. Opsin, S-Ag and cGMP were found mainly in rod cell outer segments. A polyclonal antiserum raised against transducin-alpha purified from rod outer segments predominantly labeled rod cells, but an antiserum against the carboxyterminal decapeptide of transducin-alpha labeled both rod and cone cells. Thus, most of these specialized molecules are present predominantly in rod cells, confirming major differences in components of the visual cycle in rods and cones.

Animals↗

PubMed1986

The NK-1.1(-) mouse: a model to study differentiation of murine NK cells.

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.

Animals↗