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Biomedical subjects

M F Good

Publications and source records attributed to M F Good.

At least 145 records · Page 8Linked to original sources
PubMed1987

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein.

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.

Amino Acid Sequence↗
PubMed1987

Human T clones reactive to the sexual stages of Plasmodium falciparum malaria. High frequency of gamete-reactive T cells in peripheral blood from nonexposed donors.

Malarial gametocytes, which are taken up by mosquitoes during a blood meal, develop in the gut of the mosquito into gametes. Gametes and gametocytes contain the target antigens of transmission-blocking immunity. Here, we show that the peripheral blood of nonexposed donors contains Plasmodium falciparum gamete-reactive T cells at frequencies ranging from 1/300 to 1/4000. Studies on long-term clones demonstrated that these cells often recognized antigens shared between gametes and asexual stage parasites or even between heterologous gametes, although it has been possible to derive a P. falciparum gamete-specific T clone. The T clones examined were T3+, T4+, T8-, and either HLA-DR- or HLA-DQ-restricted. They responded to gametes by both proliferation and the secretion of gamma-interferon. The gamete-specific clone and other asexual cross-reactive clones examined could be stimulated in vitro by a preparation of mature gametocytes within RBC, but not by RBC alone, suggesting that gametocytes are immunogenic or can become immunogenic for T cells in vivo. The significance of these observations to mosquito transmission of malaria and development and application of a gamete vaccine are discussed.

Adult↗
PubMed1987

In vivo testing of subunit vaccines against malaria sporozoites using a rodent system.

To test the putative in vivo protective effects of antibodies to circumsporozoite (CS) protein repeats against malarial infection, different strains of mice were immunized against various repetitive regions of the Plasmodium yoelii CS protein in the form of synthetic peptides conjugated to keyhole limpet hemocyanin. Complete Freund's adjuvant or saponin was used as adjuvant. When vaccinated mice were challenged with 500 sporozoites almost all animals became infected. There were no significant protective effects in vaccinated versus unvaccinated mice. Furthermore, there was no correlation between the antibody titer to the CS repeats and infection. The parasites from infected animals were shown to encode a CS protein containing the same repeats as those used for immunization, indicating that the infections were not due to selection for variant parasites. These experiments demonstrate that antibodies to the CS repeats, as derived in vivo with peptides, despite being surface reactive, do not provide protection against sporozoite challenge in vivo. This conclusion is in contrast to previous conclusions based on studies showing protection by way of in vitro sporozoite neutralization procedures and passive transfer of monoclonal antibody.

Animals↗
PubMed1987

Protein antigenic structures recognized by T cells: potential applications to vaccine design.

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic structures. An algorithm to search for potential amphipathic helices from sequence information identified 18 of 23 known immunodominant T-cell epitopes from 12 proteins (p less than 0.001). Another statistical approach confirmed the importance of amphipathicity and also supported the importance of helical structure that had been proposed by others. It suggested that peptides able to form a stable secondary structure, especially a helix, more commonly formed immunodominant epitopes. We used this approach to predict potential immunodominant epitopes for induction of T-cell immunity in proteins of clinical relevance, such as the malarial circumsporozoite protein and the AIDS viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens↗
PubMed1987

The effect of experimental iron-overload on splenic T cell function: analysis using cloning techniques.

The effect of iron-overload on cell-mediated immunity was examined in C57 mice. Two methods of iron-loading were used: (i) dietary carbonyl iron which produced iron-loading primarily of parenchymal cells or (ii) intraperitoneal administration of iron-dextran which produced iron-loading predominantly of Kupffer cells. Both methods of iron-loading resulted in a diminished capacity of spleen cells to generate an allo-specific cytotoxic response in the absence of exogenous interleukin 2 (IL-2). Exogenous IL-2, however, restored the ability of spleen cells from iron-loaded mice to generate allo-specific cytotoxicity in bulk culture. Clonal assays for the precursor cells of cytotoxic T lymphocytes (CTL-P), performed in the presence of added IL-2, demonstrated that iron-loaded mice contained normal numbers of CTL-P. However, cultures of spleen cells from carbonyl iron-loaded mice generated less IL-2 following Concanavalin A stimulation, apparently as a result of a reduction in the number of IL-2-secreting cells amongst the spleen cell population. This work presents further evidence that iron-overload is associated with defective immunoregulatory control.

Animals↗
PubMed1987

The effect of non-transferrin-bound iron on murine T lymphocyte subsets: analysis by clonal techniques.

A number of different immunological properties have been attributed to iron (Fe3+ and Fe2+) and iron-binding proteins. However, in many previous studies, high concentrations of iron were used and cell-cell interactions were not excluded as a possible cause of the observed immunomodulatory effects. In this study, clonal techniques have been used to examine the effect of non-transferrin-bound iron (Fe3+) on the T lymphocyte subsets required for the generation of cytotoxic T lymphocytes (CTL). Concentrations of non-transferrin-bound Fe3+ of 10 microM or greater were shown to inhibit the generation of C57, BALB/c and CBA allo-specific CTL in bulk culture. Limit-dilution analysis revealed that: (i) Fe3+ reduced the cloning efficiency of CTL-precursors (CTL-P) by up to 96% without affecting the rate of clone growth; (ii) Fe3+ did not affect the cloning efficiency of allo-stimulated Ly-2-ve T cell precursors but reduced the rate of clone growth of these cells; (iii) Fe3+ enhanced, by more than 13-fold, the function of clones of Concanavalin A (Con A)-induced suppressor T lymphocytes (STL) which suppressed in vitro the development of CTL from their precursor cells. The data provide further evidence that low concentrations of non-transferrin-bound Fe3+, of the same order as those reported to be present in the serum of patients with iron-overload, have significant immunoregulatory properties.

Animals↗
PubMed1986

Research toward malaria vaccines.

Malaria exacts a toll of disease to people in the Tropics that seems incomprehensible to those only familiar with medicine and human health in the developed world. The methods of molecular biology, immunology, and cell biology are now being used to develop an antimalarial vaccine. The Plasmodium parasites that cause malaria have many stages in their life cycle. Each stage is antigenically distinct and potentially could be interrupted by different vaccines. However, achieving complete protection by vaccination may require a better understanding of the complexities of B- and T-cell priming in natural infections and the development of an appropriate adjuvant for use in humans.

Antigens, Protozoan↗
PubMed1986

Genetic control of the immune response in mice to a Plasmodium falciparum sporozoite vaccine. Widespread nonresponsiveness to single malaria T epitope in highly repetitive vaccine.

Different H-2 congenic strains of mice were immunized with a P. falciparum sporozoite vaccine currently being tested in humans, or with different segments of the vaccine molecule. Specific IgG production or lymph node cell proliferation in response to different antigens was then determined. Only four of seven strains (representing three of eight possible different class II restriction molecules) responded to the vaccine. Of those restriction molecules, only one, I-Ab, was associated with a response to a malaria-encoded T epitope [contained within NP(NANP)3NA], while the other two molecules (E alpha dE beta d and E alpha kE beta s) were associated with a T cell response to a nonmalarial epitope(s) carboxyterminal to the malaria sequence and encoded by a tetracycline resistance gene, read out of frame. If an analogous situation applies in humans, natural boosting by sporozoites will be very restricted. This has serious implications for the effectiveness of the vaccine, since constant high levels of antisporozoite antibodies and possibly antibody-independent T cell effector functions are required for immunity.

Animals↗
PubMed1986

The effect of iron (Fe3+) on the cloning efficiency of human memory T4+ lymphocytes.

The effect of iron (Fe3+) and normal human liver ferritin on the proliferative response of normal human lymphocytes to tetanus toxoid was examined. This proliferative response involved memory T4+ lymphocytes as shown by a selective depletion study. Limit dilution analysis revealed that iron, present as ferric citrate, affected the initiation of clone development, and that concentrations of ferric citrate from 30 microM to 1 nM were able to reduce significantly the cloning efficiency of precursor T cells (up to 90% reduction). The reduced cloning frequency was not due to immunological suppression. Clone size was also reduced when iron was present during culture. In contrast, the presence of normal human liver ferritin during culture (concentration range: 300 micrograms/1-10,000 micrograms/1) had no effect on lymphocyte proliferation. The data indicate that low molecular weight iron (as ferric citrate) in concentrations similar to those which have been reported in the serum of patients with iron overload diseases, can interfere with antigen-specific lymphocyte responses and this may have implications for the development of infections and neoplasia in diseases of iron-overload.

Antigens, Surface↗
PubMed1985

A method for analysing the clonal precursors of concanavalin A-induced suppressor cells.

Spleen cells from 3 different strains of mice (C57 (H-2b), CBA (H-2k) and BALB/c (H-2d] were stimulated in vitro with different concentrations of concanavalin A (CA) for 48 h. This resulted in the production of cells capable of inhibiting the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) in a mixed lymphocyte culture (MLC). 1 microgram/ml was an effective concentration of CA to induce C57 and BALB/c suppressor cells (SC), but 5 micrograms/ml CA was required to induce CBA SC. SC precursors (SC-P) were shown to be radiosensitive and the results suggest that SC themselves may be radiosensitive. SC were effective in the presence of added interleukin-2 (IL-2). SC were then induced at limit dilution in microwells in a volume of 25 microliter. A MIC (200 microliter) was then (after 48 h) added to each microwell. This resulted in a dilution of the concentration of CA to a level below which it was effective at inducing suppression. Cytotoxicity was then assessed 7 days later. It was thus possible to analyse SC-P at the clonal level and estimate their frequency. The frequency of C57 splenic SC-P (active against a C57 anti-BALB/c MLC) was 14.4 X 10(-6), the frequency of CBA splenic SC-P (active against a CBA anti-BALB/c MLC) was 92.3 X 10(-6), and the frequency of BALB/c splenic SC-P (active against a BALB/c anti-CBA MLC) was 15.8 X 10(-6). It was possible to analyse SC-P at a clonal level whether or not the MLC contained added IL-2. SC and SC-P were shown to be sensitive to anti-Thy-1 and complement.

Animals↗
PubMed1984

Activated lymphocyte killer cells derived from melanoma tissue or peripheral blood.

Lymphoid cells infiltrating metastatic melanomas were grown directly from cell suspensions of tumour tissue by the addition of T cell growth factor. Lymphoid cells grew out at the expense of tumour cells in six of seven freshly excised tumours, and cells from two cultures were expanded for in vitro testing of cytolytic function against different target cells. Early in culture the tumour derived lymphocytes killed fresh autologous melanoma cells and, particularly later in culture, were highly and non-specifically cytolytic for cultured melanoma and non-melanoma cells. Cultured peripheral blood lymphocytes from patients with melanoma, and from normal subjects, were cytolytic to the same degree as tumour derived lymphocytes, and also resembled cells grown from tumour tissue in possessing acid phosphatase activity which was resistant to tartrate. Cultured lymphoblasts from both tumour and peripheral blood had a T cell phenotype when analysed with monoclonal antibodies. An in vitro co-culture system was employed to study the kinetics and the precursors of these non-specific killer cells among blood mononuclear cells. Blood mononuclear cells cultured with irradiated B lymphoblasts led to the generation of non-specific cytolytic cells, referred to as activated lymphocyte killer (ALK) cells, after 7-10 days of culture and the progenitors of these ALK cells were demonstrated to be distinct from those of specific cytolytic T cells.

Cells, Cultured↗
PubMed1983

Functional clonal deletion of cytotoxic T-lymphocyte precursors in chimeric thymus produced in vitro from embryonic Anlagen.

Chimeric thymus, formed by fusing the prelymphoid third pharyngeal pouches of fetal mice with fetal liver, have been allowed to develop entirely in vitro. Syngeneic and allogeneic chimeras were prepared and both types of thymus were shown to contain substantial numbers of functional cytotoxic T lymphocyte precursors reactive against "third party" alloantigens. However, alloreactivity specific for H-2 antigens present on either the third pharyngeal pouch or the fetal liver was minimal. In three different allogeneic chimeric thymuses, the frequencies of cytotoxic T lymphocyte precursors reactive to H-2 antigens present on the third pharyngeal pouches were reduced to 1%, 4%, and 0% of control values, whereas, in the one allogeneic chimera tested for alloreactivity to H-2 antigens present on the fetal liver, the cytotoxic T lymphocyte precursor frequency was reduced to less than 1% of control values. The phenotype of the H-2 tolerance is shown to be one of functional clonal deletion of the cytotoxic T lymphocyte precursor.

Animals↗
PubMed1983

Clones of cytotoxic T lymphocytes reactive to haptenated allogeneic cells: precursor frequency and characteristics as determined by a split-culture approach.

CBA (H-2k) responder spleen cells have been cultured at limit dilution with trinitrophenyl (TNP)-modified BALB/c (H-2d) stimulator cells and a source of T-cell growth factor in order to generate cytotoxic effector clones. After culture, such clones were split into two to four replicates and each was assayed against a different target. This allowed identification of clones capable of lysing TNP-modified P815 (H-2d) targets but not unmodified P815 targets. Thus, clones specific for TNP and allogeneic restriction elements were detected without the need to use techniques that deplete the responder population of alloreactive cells. Cytotoxic T lymphocyte precursors (CTL-P) specific for TNP-modified P815 (major histocompatibility complex-nonidentical) targets were identified, at a low frequency (28.2 x 10(-6)) compared to CTL-P for TNP-modified C1.18 (H-2k) (identical) targets (224 x 10(-6)). The hapten specificity, H-2 restriction specificity, and Thy-1 status of these clones have been examined. Fourteen percent of CBA CTL-P reactive to TNP-modified P815 targets also showed reactivity to NIP-modified P815 targets, and 86% of CBA clones reactive to TNP-modified P815 targets ("allo-TNP-reactive" clones) failed to show reactivity to TNP-modified C1.18 targets--i.e., showed a restriction preference for allo rather than self. All such H-2d-restricted, TNP-specific clones were uniformly sensitive to anti-Thy-1 antibody and complement. Among the H-2k responders studied, we have not demonstrated CTL-P reactive to TNP-modified syngeneic cells which also react with H-2d cells or NIP-modified H-2d cells among 168 clones analyzed. This suggests that such clones, if present, are relatively rare.

Animals↗
PubMed1983

Analysis of true anti-hapten cytotoxic clones in limit dilution microcultures after correction for "anti-self" activity: precursor frequencies, Ly-2 and Thy-1 phenotype, specificity, and statistical methods.

By the use of split culture techniques we have been able to demonstrate conclusively that the "anti-self" activity and the spontaneous anti-hapten activity within an apparent anti-hapten cytotoxic T cell response is a clonal phenomenon and is not caused by cross-reactivity between anti-self and true anti-hapten clones. With the knowledge of this clonality we have been able formally to prove that the true hapten-generated, hapten-specific response can be obtained by subtracting the response generated by "self" but directed against modified targets from the response generated by modified self and directed against modified targets. Unbiased statistical estimators, which do not make assumptions about the origin (0 responder cells, 100% negative cultures), have been developed to plot accurately limit dilution data (maximal likelihood estimator and minimal chi-square estimator), and the analysis demonstrates that all the components of an apparent anti-hapten response obey zero order (single hit) kinetics. By specifically identifying true anti-hapten and true anti-self clones, we have been able to study the phenotype of their precursors as well as the effectors themselves at the clonal level. Precursors of true anti-hapten and anti-self clones are Thy-1+, Ly-2+. However, anti-hapten and anti-self effector cells show marked clonal variation with respect to Ly-2, as some clones are almost completely insensitive to anti-Ly-2 and complement whereas others show minimal to complete sensitivity. All anti-hapten clones are completely sensitive to anti-Thy-1 and complement, whereas about one-third of anti-self clones show only partial sensitivity, with the most lytic clones showing the most sensitivity. Hapten-specificity and anti-self-specificity have been examined clonally. Ten percent of anti-TNP clones recognize NIP, 10% of anti-NIP clones recognize TNP, and 20% of anti-self clones recognize an allo-target. These figures are in accordance with the overall specificity of effectors generated in bulk culture in the presence of CAS (concanavalin A-stimulated spleen cell conditioned medium). However, hapten specificity and H-2 restriction of bulk generated effectors are improved if CAS is omitted from cultures.

Animals↗
PubMed1983

Functional clonal deletion and suppression as complementary mechanisms operative in adult hapten-induced cytotoxic T cell tolerance.

Immunologic tolerance to the hapten TNP was induced in adult mice through the i.v. injection of reactive TNBS. To probe the cellular basis of the tolerant state, splenic cytotoxic T lymphocyte precursors (CTL-P) were stimulated in vitro with haptenated, x-irradiated syngeneic spleen cells in the presence or absence of exogenously added growth factors derived from Concanavalin A-stimulated spleen cell conditioned medium (CAS). The cultures were either conventional bulk cultures or limit dilution cloning cultures. For the latter, cytotoxicity was assessed through a semi-automated, radioautographic 111In-release assay. Suppressive potential was assessed by mixing spleen cells from tolerant mice with normal spleen cells before culture. In the absence of CAS, bulk cultures showed profound tolerance, and suppressive capacity was clearly evident. Suppression was dependent on the presence of TNP-self during culture and affected the generation of CTL from CTL-P and not the effector function of CTL. Cyclophosphamide treatment did not prevent tolerance induction. In the presence of CAS, bulk cultures still showed marked tolerance, but mixing experiments now yielded no evidence of suppression. As documented previously, limit dilution cultures of tolerant spleen cells in the presence of CAS showed a functional clonal deletion of hapten-specific CTL-P. In the absence of CAS, limit dilution cultures became dependent on helper T cells as the limiting element. Tolerant populations showed a diminution of activatable helper T lymphocyte precursors (HTL-P), which may have been due to a functional clonal deletion of HTL-P and/or a concomitant activation of suppressor T cells. Adoptive transfer studies showed that cells from tolerant mice did not detectably influence the number of hapten-specific CTL-P in the spleens of host animals. Taken together, the results suggest that both functional clonal deletion of CTL-P and suppression of HTL-P contribute to the tolerant state induced.

Animals↗
PubMed1983

Characteristics of tolerance induction among adult hapten-specific T lymphocyte precursors revealed by clonal analysis.

Thaptens 2,4,6-trinitrobenzene sulfonic acid (TNBS) and 3-iodo-4-hydroxy-5-nitrophenyl-acetic acid succinamide ester (NIP) on murine splenic and thymic cytotoxic T lymphocyte precursors (CTLp) was studied. Both BALB/c (H-2d) and CBA (H-2k) CTLp can be rendered unresponsive to subsequent in vitro stimulation with either hapten, provided the mice were pretreated with the correct dose of reactive hapten. The unresponsiveness was shown not to be due to toxicity, because treated animals can mount an alloreactive response equivalent to untreated animals. A degree of cross-reactivity in tolerance induction, however, was demonstrated between the two different haptens. Limit dilution analysis of tolerant lymphoid populations reveals that such cells obey single hit (zero order) kinetics and that there is effective deletion of the CTLp. After correction for "anti-self" responses, the anti-TNP CTLp frequency in TNBS-treated BALB/c mice was 10.7 X 10(-6) in contrast to 101.1 X 10(-6) in control animals (89.4% tolerance). Similarly, after correction for "anti-self" responses, the anti-NIP CTLp frequency in NIP-treated animals was 4.7 X 10(-6), in comparison to 42.8 X 10(-6) in control animals (89.0% tolerance). Kinetic studies show that tolerance occurs within 24 hr. This, together with the limit dilution data, argues against the role of suppressor cells, over the cellular range studied.

Animals↗
PubMed1982

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity.

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.

Animals↗