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Biomedical subjects

C J Hackett

Publications and source records attributed to C J Hackett.

At least 37 records · Page 2Linked to original sources
PubMed1985

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus.

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.

Amino Acid Sequence↗
PubMed1985

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins.

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.

Animals↗
PubMed1985

Influenza-specific suppression: contribution of major viral proteins to the generation and function of T suppressor cells.

Suppressor cells were generated in BALB/c mice by two sequential injections of PR8 influenza virus (A/Puerto Rico/8/34[H1N1]) and were tested for their ability to inhibit proliferative cellular responses towards multiple viral and nonviral antigens. In this way, suppression specific to PR8 as compared with purified protein derivative (PPD) and keyhole limpet hemocyanin (KLH) antigenic responses was illustrated. Experiments involving adoptive transfer of suppression to naive hosts with subfractionated lymphocyte populations demonstrated that the suppressors were Lyt-2+ T cells. Two major questions were addressed with this system. First, a determination was made of which anti-viral protein proliferative responses were affected by the PR8-induced T suppressor (Ts) cells. Ts cells were found to inhibit proliferating cells with specificities for isolated hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix (M) antigens. Second, experiments were conducted to analyze the viral proteins contributing to the induction of PR8-specific Ts cells. Inoculations with either isolated HA or a combination of M + NP proteins induced T suppression specific to proliferative responses towards PR8. These experiments illustrate the contribution of external (HA and NA) as well as internal (M + NP) viral proteins to Ts cell generation and function.

Animals↗
PubMed1984

On the epidemiology of yaws in African miners (1942).

Several points of interest arise from these unusual human yaws infections: (i) European miners contracted a treponemal infection from Africans with yaws and developed yaws; (ii) the infectious patients who started the minor epidemics in the outbreaks must have been relapses from prolonged latent early infections; (iii) the time taken for the treponemes, possibly only a few minutes, to penetrate the skin through a minor injury seemed to be less than six hours; (iv) transmission occurred only at the working rock face deep underground with a tropical climate, and not on the surface 1,630 metres above sea-level, where there were abundant opportunities; (v) the search for treponemes in the environment of the working place was restricted by the inability to culture the treponeme.

Adolescent↗
PubMed1984

Characterization of the murine TH response to influenza virus hemagglutinin: evidence for three major specificities.

Several hemagglutinin (HA)-specific T cell hybridomas were generated from five PR8 influenza virus-primed BALB/c mice by fusion with the thymoma BW5147 and were tested for IL 2 (interleukin 2) production in response to a large panel of serologically distinct influenza viruses and HA-protein fragments. The hybridomas (provisionally categorized in the helper T cell lineage) could be placed into three major specificity groups based on their reactivity patterns toward 43 antibody-selected PR8 virus mutants that had been shown previously to differ from parental PR8 virus by defined (usually single) amino acid substitutions in the HA1 polypeptide. Three hybridomas with specificity 1 failed to respond to the PR8 mutant RV6 (Glu115----Lys), two with specificity 2 failed to respond to PR8 mutant DV4 (Ser136----Pro), and two with specificity 3 responded to all antibody-selected PR8 mutants without exception. The three reactivity patterns could be used along with further biochemical evidence to demonstrate the presence of three distinct T cell determinants on the HA1 polypeptide. Priming of mice with one of these T cell determinants in the form of a synthetic peptide induced a T cell subset able to proliferate in vitro in response to HA and intact PR8 virus. Further analysis of the T cell hybridomas for reaction with 17 natural influenza virus isolates of the H1 subtype revealed additional differences in fine nominal antigen specificity among these T cells. However, two pairs of T cells whose members were isolated from different mice exhibited indistinguishable fine nominal antigen specificities. The latter analysis showed that natural antigenic drift (i.e., accumulation of point mutations) in the HA occurring between the years 1934 and 1957 and again between 1977 and 1980 altered two T cell determinants but left one T cell determinant unchanged. The shifts from H1 to H2 or H3 subtypes resulted in the alteration of all three T cell determinants. The present findings suggest that 1) immunization with influenza virus induces HA-specific T cells that recognize determinants different from those seen by anti-HA antibodies, and 2) the HA-specific T cell repertoire of adult virus-primed BALB/c mice is more restricted than the B cell repertoire.

Animals↗
PubMed1983

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

Amino Acid Sequence↗
PubMed1983

Stimulation of cytotoxic T cells by liposomes containing influenza virus or its components.

Since inactivated virus preparations are poor inducers of influenza-specific cytotoxic T cells (Tc), studies were undertaken utilizing artificial vesicles (liposomes) as a means of delivering viral and H-2 antigens in a multivalent form and oriented with respect to a lipid bilayer. Liposomes prepared from extracted mouse cell lipids efficiently incorporated influenza-viral proteins and were not toxic in culture. Using polybrene to promote greater contact of liposomes with cells, liposomes prepared from whole virus could effectively stimulate memory Tc from spleens of intranasally infected mice in vitro. H-2 was not required in the liposomes to obtain stimulation, and its presence did not improve responses, which were always lower than in parallel stimulations using virally infected syngeneic cells. Liposomes prepared from purified influenza virion subunits (haemagglutinin, neuraminidase, matrix protein) were only slightly stimulatory in vitro, and were unable to prime mice for significant Tc memory.

Animals↗
PubMed1982

A convenient culture chamber for observation and embedding of macrophage monolayers for transmission electron microscopy.

A chamber adapted from a polypropylene test tube is described for the cultivation and processing of undisturbed monolayers of normal mouse macrophages for transmission electron microscopy (TEM). Peritoneal exudate cells are grown in the chambers on Visking dialysis membranes pretreated with Polybrene (Sigma Chemical Co.) and normal mouse serum. The cells are not further disturbed after adherence since the entire chamber, including dialysis membrane and cells, is processed for electron microscopy using standard material and protocols and the embedded dialysis membrane is readily oriented and thin-sectioned. This method is efficient for studying adherent cells since growth can be nearly confluent and cells remain firmly attached throughout the manipulations. Since a transparent membrane is used, cells can also be observed with the light microscope at all stages prior to sectioning.

Animals↗
PubMed1982

Evidence for two T-helper populations with distinct specificity in the humoral response to influenza A viruses.

Virus specificity of T-helper cells for the humoral antibody response to influenza A viruses was studied with a hapten-carrier secondary adoptive transfer system, using whole virus, or viral components inserted into liposomes as carrier with B cells primed to DNP human gamma globulin. Evidence was obtained for two distinct T-helper cell populations from mice primed by influenza infection: a T-helper cell cross-reactive for all type A influenza viruses and a second T-helper population specific for the variant haemagglutinin. In vivo the virus cross-reactive T helpers recognized whole virus, but did not recognize isolated surface glycoproteins or internal virus proteins.

Animals↗
PubMed1982

H-2 and viral haemagglutinin expression by influenza-infected cells; the proteins are close but do not cocap.

The proximity of H-2K and D antigens and influenza virus haemagglutinin (HA) molecules on the surface of infected target cells was assessed by a topographical study using monoclonal antibodies to H-2 and to HA. The effect of pretreatment of fixed, infected cells with excess of one monoclonal antibody on the subsequent binding of a second radiolabelled antibody was measured. Using CBA mouse B lymphoblasts which were paraformaldehyde fixed 5 hr postinfection with influenza virus (A/USSR/90/77), pretreatment with monoclonal antibody 30/3 to H-2Kk and Dk partially blocked (Approximately equal to 37%) the binding of one radiolabelled monoclonal anti-HA antibody (264/2). A different monoclonal IgG (W18/1) directed to the same HA molecule was not blocked by similar pretreatment of cells with the anti-H-2 antibody. Interaction of monoclonal antibodies with their sites is highly specific, and mutual blocking of two antibodies requires very closely located sites even if the antibodies are directed to the same molecule. We therefore have evidence for proximity of H-2 and HA molecules; however, we were unable to demonstrate cocapping of H-2K and D antigens with influenza HA.

Animals↗
PubMed1981

H-2 expression by lymphoid cells of different mouse strains: quantitative interaction of H-2 with monoclonal antibodies and their Fab fragments.

Monoclonal antibodies (H100-30/3 and 11-4.1) to H-2k were used to study H-2 antigen expression and characteristics of the H-2 antigen-antibody interaction at the cell surface. Studies with radiolabelled F(ab')2 and Fab' fragments of 11-4.1 antibody confirmed that monoclonal IgG binding to cells is directly proportional to the number of H-2 sites and shows a high proportion of monovalent binding over a wide range of concentrations. Scatchard plots showed no difference in the binding affinity constant (Ka) of a given monoclonal antibody on lymphoblasts from various H-2k F1 and congenic strains, but only in the number of antigenic sites per cell. F1 (k x d) lymphoblasts show 1 x 10(5) H-2k sites/cell, about 50% of the expression in homozygotes. Dk expression in C3H.OH is 1.4 x 10(4) sites/cell. While normal cells appear to have a constant amount of H-2 (2-3 x 10(5) sites/cell), BW thymoma cells show unstable H-2 expression, having an average of five times fewer H-2 sites per cell when grown in vitro as compared to in vivo growth. Another BW cell surface marker, Thy-1.1, does not fluctuate in parallel with H-2. The 30/3 and 11-4.1 antibodies bind to topologically distinct sites on H-2Kk. The binding of these antibodies can be perturbed differentially: paraformaldehyde fixation of cells abolishes binding of 11-4.1 antibody but not of 30/3 antibody; increasing temperature increases the Ka of 30/3 antibody binding but decreases the Ka of 11-4.1 antibody binding to cells.

Animals↗
PubMed1980

Quantitation of influenza virus antigens on infected target cells and their recognition by cross-reactive cytotoxic T cells.

Monoclonal antibody to type-A influenza virus matrix (M)-protein was used to quantitate the appearance of M-protein on abortively infected P815 cells. After 16 h of infection with different type-A viruses, only a low amount of M-protein appears on the surface of infected cells (approximately 10(3) site/cell) in contrast to approximately 10(5) hemagglutinin molecules on each cell surface. However, virus replication is required for M-protein appearance. Analysis of solubilized membranes purified from 16-h-infected cells shows approximately 10(4) M-protein molecule/cell in the plasma membrane, a content that is consistent with the observed low surface expression, and that indicates that most of the M-protein is localized internally. We found no evidence that cross-reactive cytotoxic T cells could recognize M-protein; neither monoclonal antibody or hyperimmune anti-M-protein antiserum could inhibit T cell killing, either alone or in combination with monoclonal anti-H-2 antibody. Taken together, the low level of M-protein appearance and lack of T cell blocking by anti-M-protein antibody leaves doubt that M-protein is the antigen recognized by cross-reactive cytotoxic T cells.

Animals↗
PubMed1980

Monoclonal antibodies to influenza matrix protein: detection of low levels of matrix protein on abortively infected cells.

We have quantified the appearance of matrix (M-) protein on P815 mastocytoma cells infected with type A influenza virus using a monoclonal antibody specific for M-protein. In contrast to previously reported values, only low amounts (about 10(3) molecules/cell) of M-protein appear on the cell surface up to 16 h p.i. Since P815 cells are excellent targets for cross-reactive T-cell lysis 3 to 5 h after virus infection, when only about 10(2) M-protein sites are found on each cell surface. It appears less likely that the recognition of M-protein can account for the cross-reactivity of cytotoxic T-lymphocytes for different type A influenza viruses.

Animals↗