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Biomedical subjects

U Krzych

Publications and source records attributed to U Krzych.

At least 37 records · Page 2Linked to original sources
PubMed1994

Immunization with irradiated Plasmodium berghei sporozoites induces IL-2 and IFN gamma but not IL-4.

Protective immunity against Plasmodium induced by immunization with irradiated sporozoites (SPZ) depends on both humoral and cellular responses. Although circumsporozoite protein (CSP)-specific cytolytic T lymphocyte responses have been established as an effector system, other cell types are required for protection. We have previously demonstrated that although protective immunity and T cell proliferative reactivity to SPZ are mouse strain- and SPZ dose-dependent, no correlation between the two responses could be found. Since protective immunity involves functionally diverse T cell subsets, we asked whether the discordance between proliferative responses to SPZ and protective immunity might have resulted from selective activation of either the Th1 or Th2 cell subset. Protective immunity, in vitro proliferative responses, and lymphokine production were tested in BALB/c, C57Bl/6, and C3H/HeN mice immunized according to different SPZ regimens. The levels of IL-2 paralleled the proliferative reactivities in each mouse strain examined. Although IFN gamma levels were present in the unprimed lymphocyte cultures, they increased following each SPZ immunization, in C57Bl/6, moderate in C3H/HeN, and lowest in BALB/c splenic cultures. Surprisingly, no IL-4 was detected in splenic cultures from any mouse strain during proliferative activity or protective immunity. In contrast, elevated IL-6 production was noted after each immunization, regardless of the protective status and it correlated with anti-CSP IgG serum levels. These data establish that lymphokine profiles corresponding primarily to the Th1 cells were induced by immunization with P. berghei SPZ and that IL-4 secreting T cells were not induced by the SPZ-stage berghei antigens.

Animals↗
PubMed1993

Plasmodium berghei-specific T cells respond to non-processed sporozoites presented by B cells.

The mechanism of malaria protective immunity induced by immunization with radiation-attenuated Plasmodium sporozoites (SPZ) is only partially understood. For example, B and T cell responses specific for the circumsporozoite (CS) protein, a 46 kDa SPZ surface protein, have been characterized; however, events leading to SPZ-specific T cell activation, i.e., processing and presentation of SPZ by antigen-presenting cells have not been investigated. In the present study we describe the in vitro analysis of requirements for accessory cell function in the presentation of SPZ to SPZ-immune T cells. The results establish that SPZ-induced proliferative T cells are reactive to non-processed SPZ presented by activated B cells and, thus, imply that the non-processed form of the SPZ-associated CS protein restricts the induction of the potential CS protein T cell repertoire.

Animals↗
PubMed1993

Induction of cytolytic and antibody responses using Plasmodium falciparum repeatless circumsporozoite protein encapsulated in liposomes.

Plasmodium circumsporozoite (CS) protein-induced antibody and T-cell responses are considered to be important in protective immunity. Since the key repeat determinant of the CS protein may actually restrict the recognition of other potential T- and B-cell sites, a modified Plasmodium falciparum CS protein lacking the central repeat region, RLF, was expressed in Escherichia coli. On purification, RLF was encapsulated into liposomes [L(RLF)] and used for the in vivo induction of cytolytic T lymphocytes (CTL) and antibodies. Immunization of B10.Br (H-2k) mice with L(RLF), but not with RLF, induced CD8+ CTL specific for the P. falciparum CS protein CTL epitope, amino acid residues 368-390. Anti-L(RLF) serum reacted with antigens on intact sporozoites and inhibited sporozoite invasion of hepatoma cells. Antibody specificity studies in New Zealand White rabbits revealed new B-cell sites localized in amino acid residues 84-94, 91-99, 97-106 and 367-375. Although the mechanisms by which liposomes enhance cellular and humoral immune responses remain unknown, liposome-formulated vaccines have been well tolerated in humans; hence, their use in vaccines, when efficacy depends on antibody and CTL responses, may be broadly applicable.

Amino Acid Sequence↗
PubMed1992

Distinct T cell specificities are induced with the authentic versus recombinant Plasmodium berghei circumsporozoite protein.

Plasmodium berghei sporozoite (SPZ)-immune lymph node (LN) cells obtained from mice of different H-2 haplotypes were analyzed for the presence of circumsporozoite (CS) protein-reactive T cells in proliferative assays. Although lymphocytes from each strain responded in vitro to the priming Ag and to the soluble rCS protein, they did not respond to CS protein synthetic peptides. Parallel analysis of rCS protein-primed LN cells revealed that the two Ag are unequal in generating T cell specificities: although SPZ priming did not induce CS protein peptide-reactive T cells, priming with rCS protein did. Not being privy to the processing and presentation of SPZ Ag, we postulated that a different order of processing of the authentic, i.e., SPZ-associated CS protein vs soluble rCS protein might be responsible for the generation of different T cell specificities. Accordingly, authentic CS protein might not be processed by APC, or the processed fragments might obscure the recognition of smaller peptide fragments. Therefore, we subjected the SPZ to three cycles of a freeze/thaw procedure and used the denatured SPZ preparation for priming. We observed that contrary to priming with the authentic SPZ, denatured SPZ generated T cells reactive to some of the CS protein synthetic peptides. The hypothesis that each form of the SPZ Ag is subject to a unique Ag processing was also confirmed in experiments demonstrating a lack of recognition of the authentic CS protein by rCS protein-primed LN cells. Hence, the evidence presented in this work that complex protozoan Ag, such as Plasmodia, might present different requirements for Ag-specific T cell induction/activation not only enhances the basic understanding of the immune system, but is essential for the development of antimalaria vaccine(s). In addition, these observations support the hypothesis that the molecular context of the priming Ag influences the outcome of T cell specificities, by providing evidence that the authentic CS protein induces a T cell repertoire that is distinct from that induced by the rCS protein.

Amino Acid Sequence↗
PubMed1992

Adjuvant effects of liposomes containing lipid A: enhancement of liposomal antigen presentation and recruitment of macrophages.

Liposomes containing lipid A induced potent humoral immune responses in mice against an encapsulated malaria antigen (R32NS1) containing NANP epitopes. The immune response was not enhanced by lipid A alone or by empty liposomes containing lipid A. Experiments to investigate the adjuvant mechanisms of liposomes and lipid A revealed that liposome-encapsulated R32NS1 was actively presented by bone marrow-derived macrophages to NANP-specific cloned T cells. The degree of presentation was related to the amount of liposomal antigen added per macrophage in the culture medium. At high cell densities, poor presentation occurred when liposomes lacked lipid A but excellent presentation occurred when the liposomes contained lipid A. Liposomes containing lipid A and encapsulated antigen also activated gamma interferon-treated macrophages to produce nitric oxide. Macrophage activation and antigen presentation occurred with liposomes that could not be detected by the Limulus amebocyte lysis assay. Intraperitoneal injection of liposomal lipid A caused a marked increase in the recruitment of immature (peroxidase-positive) macrophages to the peritoneum. On the basis of these experiments, we propose that the mechanism of the adjuvant action of liposomal lipid A is partly due to increased antigen presentation by macrophages and partly due to recruitment of an increased number of macrophages serving as antigen-presenting cells.

Adjuvants, Immunologic↗
PubMed1991

Use of adjuvant containing mycobacterial cell-wall skeleton, monophosphoryl lipid A, and squalane in malaria circumsporozoite protein vaccine.

Human immune responses to modern synthetic and recombinant peptide vaccines administered with the standard adjuvant, aluminum hydroxide, tend to be poor, hence the search for better adjuvants. Antibody responses to a Plasmodium falciparum circumsporozoite (CS) protein vaccine, R32NS1(81), administered with an adjuvant containing cell-wall skeleton of mycobacteria and monophosphoryl lipid A in squalane (MPL/CWS) have been compared to responses to the same immunogen administered with aluminum hydroxide. 2 weeks after the third dose the following indices were greater in the 5 patients who received MPL/CWS than in controls (p less than 0.05): the geometric mean concentration (2.0 vs 25.4 microgram/ml) and avidity index of antibodies to the P falciparum CS protein by ELISA, the geometric mean titre to P falciparum sporozoites by IFAT (1/115 vs 1/1600), and the geometric mean inhibition of sporozoite invasion of hepatoma cells in vitro (37.6 vs 90.3%). For R32NS1(81) MPL/CWS is superior to aluminum hydroxide as an adjuvant, and the data support the evaluation of this complex as an adjuvant for other vaccines.

Adjuvants, Immunologic↗
PubMed1991

The distinctive specificity of antigen-specific suppressor T cells.

Although suppressor T cells have been cloned in only a few instances, the existence of a functional cadre of T cells that acts to downregulate the immune response is well documented. In this review Eli Sercarz and Urszula Krzych describe studies on suppressor T-cell (TS-cell) specificity that provide some support for the conclusion that the TS cell is a distinctive cell type with an expressed repertoire that is different from that expressed by helper T (TH) cells. They go on to explore the interaction between cells recognizing TS-cell-inducing determinants (SDs) and TH-cell-inducing determinants (HDs), and their relationship to immunogenicity and Ir gene effects.

Amino Acid Sequence↗
PubMed1990

Immune response to Plasmodium berghei sporozoite antigens. I. Evaluation of murine T cell repertoire following immunization with irradiated sporozoites.

The Plasmodium berghei sporozoite antigen-specific T cell repertoire was analyzed in C57BL/6 (H-2b), BALB/c (H-2d) and C3H/HeN (H-2k) mice following immunization with irradiated sporozoites. Proliferative responses were correlated with the protective status of each strain. Proliferative reactivities to sporozoite antigens were compared in cultures containing either CD4+ T cells, CD8+ T cells, or total splenic lymphocytes. CD8+ T cells had no proliferative activity to sporozoite antigens; CD4+ T cells and splenic lymphocytes responded to the priming antigen, but the responses varied according to the mouse strain tested. The proliferative activity diminished at the onset of protection, presumably due to the induction of regulatory or non-proliferative T cell subsets. Sporozoite-immune lymphocytes did not respond to P. berghei circumsporozoite synthetic peptides. The restricted utilization of T cell epitopes during anti-sporozoite responses can be interpreted as resulting in part from a limited processing of the CS protein antigen.

Amino Acid Sequence↗
PubMed1990

Role of circumsporozoite protein-specific T-cells in protective immunity against Plasmodium berghei.

The Plasmodium berghei circumsporozoite (CS) protein-specific T-cell repertoire was analysed in C57Bl/6 (H-2b), Balb/c (H-2d), and C3H/HeN (H-2k) mice immunized with irradiated sporozoites and the proliferative responses were correlated with the protective status of each strain. Splenic lymphocytes responded to the priming antigen, but the responses varied according to both murine strain and immunization schedule. Analysis of cytolytic T lymphocyte (CTL) responses in mice immunized with irradiated P. berghei sporozoites or with a Salmonella-recombinant CS protein construct revealed that each immunization induces CTLs recognizing different epitopes on the target cells. The variations in immune reactivities among different murine strains to the CS protein antigens and the variations in the responses to the authentic versus recombinantly expressed CS protein suggest that distinct immune mechanisms may be involved in rendering immune protection. Furthermore, the molecular context of the immunizing antigen may influence the outcome of the fine specificity of T cells involved in immune protection.

Animals↗
PubMed1989

Sequences outside a minimal immunodominant site exert negative effects on recognition by staphylococcal nuclease-specific T cell clones.

In recent years, synthetic peptides have been utilized extensively to characterize the minimal essential immunodominant sites on model protein Ag. However, little work has focused on the effect that sequences flanking these minimal recognition sites may exert on T cell recognition. Previous work with staphylococcal nuclease (Nase) demonstrated that I-Ek-restricted clones recognize the peptide 81-100, whereas I-Ab-restricted clones recognize the over-lapping but non-cross-reacting peptide 91-110. Further analysis with 15 or 10 residue peptides within the region 81-110 reveals that the minimal sequence capable of stimulating I-Ek-restricted clones is contained within the decapeptide 91-100. Addition of residues 86-90, to give the peptide 86-100, enhanced the recognition substantially, whereas addition of residues 101-105 produced a 91-105 peptide with no stimulatory ability. These results suggest that interactions between the antigenic peptide 91-100 and residues within the flanking 101-105 sequence have negative consequences for presentation of the immunodominant epitope to T cell clones. Introduction of single amino acid substitutions within 91-105 produced peptides that induce responses comparable to those seen with 91-100. These results are consistent with the suggestion of negative interactions between the minimal immunodominant site and flanking sequences in that single residue substitutions may remove these negative interactions and lead to restoration of stimulatory ability. The negative effect of flanking sequences on T cell recognition of immunodominant sites presents new considerations for development of synthetic vaccines as well as for understanding the biology of Ag processing and presentation.

Amino Acid Sequence↗
PubMed1989

The molecular context of determinants within the priming antigen establishes a hierarchy of T cell induction: T cell specificities induced by peptides of beta-galactosidase vs. the whole antigen.

The antibody response following priming with a macromolecule or a peptide will depend on the regulatory T cells that become activated by the antigenic determinants available. In this report, activation of T helper (Th) and T suppressor (Ts) cells by determinants on beta-galactosidase (GZ) was examined by comparing native GZ [1023 amino acid (a.a.) residues per monomer] with peptides from the immunodominant region encompassing residues 3 to 187. Each immunogen established its characteristic hierarchy of dominance of determinants within it: in particular, GZ and CB-2-3 (a.a. 3-187) each induced immunodominant Th cells which could not be induced by T8 (a.a. 60-140). Hierarchies of suppressor determinant are also created: T8-Ts suppresses all Th specificities and therefore can be deemed immunodominant: T8-2-Ts and T8-3-Ts have a more selective suppressor activity and can be considered subdominant. We conclude that the outcome of immunization shifts with a change in the nature of the immunogen and the context within which the determinant lies will crucially influence its expression. A particular "context" presumably determines the likely order of processing of that molecule which leads to a characteristic relationship among the Ts, Th and B cell determinants involved.

Animals↗
PubMed1988

Induction of helper and suppressor T cells by nonoverlapping determinants on the large protein antigen, beta-galactosidase.

The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-inducing determinants was examined with cyanogen bromide and tryptic peptides of Escherichia coli beta-galactosidase (GZ), a large tetrameric protein (monomer molecular weight = 116 kDa). Immunization with cyanogen bromide fragment 2 [CB-2, amino acids (a.a.) 3-92] induced both specific Th and Ts cells. Study of the induction of these functionally opposite T cell subpopulations with tryptic peptides of CB-2 indicated that Th and Ts were activated by separate, nonoverlapping determinants. Th-inducing activity resided in a nonapeptide, T6 (a.a. 44-52), whereas T4 (a.a. 27-37) induced Ts cells. The presence of distinct helper and suppressor determinants suggests that the specificity repertoire in these T cell subpopulations may differ, perhaps owing to the expression of antigen-recognizing receptors that are coded by unique gene families. Alternatively, antigen presentation structures may be physicochemically quite different, and bind to distinct parts of the peptide antigen.

Animals↗
PubMed1986

The relationships between Ts-inducing and Th and Tp-inducing determinants on a large protein antigen.

The immunogenicity of several small monomeric protein antigens - lysozyme, myoglobin, cytochrome c and insulin - has been intensely studied during the past decade to try to learn the rules of the game. It is likely that before the stage is reached at which we can predict the nature of the determinants responded against by each lymphoid subpopulation, other types of molecules such as fibrous proteins, multimeric large proteins and viral capsid proteins will have to be examined, and such studies have commenced recently. In an effort to generalize our studies with lysozyme to one of these systems, we have been exploring the response of H-2k, CBA/J mice to E. coli B-galactosidase, GZ, a tetramer with a monomer molecular weight of 116,250. We feel that aspects of this system have important implications for the vaccine problem, and these will be summarized at the end of the article.

Animals↗
PubMed1985

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. II. Only certain T helper or T suppressor cells are relevant in particular regulatory interactions.

11 cyanogen bromide (CB) peptides, comprising 70% of the large protein, Escherichia coli beta-galactosidase (GZ), were studied for their ability to induce T suppressor (Ts) cells capable of strongly suppressing the in vitro anti-fluorescein (FITC) response to GZ-FITC. Only CB-2 (amino acid residues 3-92) and CB-3 (residues 93-187) were found to bear such Ts-inducing epitopes. In examining the specificity of T helper cell (Th) targets susceptible to CB-2 and CB-3-specific Ts, it appeared that only nearly Th targets could be suppressed. Thus, CB-10-primed Th were not suppressed by either Ts; even CB-3-primed Ts did not suppress CB-2-specific Th, although CB-2-specific Ts were effective. Furthermore, analysis of the suppression pattern revealed a hierarchical use of potential epitopes on native GZ in triggering functional regulatory T cells. A dominant Th epitope near the amino terminus of GZ tops a hierarchy of potential Th, most of which are never engaged. The dominant determinant seems to exist on the peptide CB-2-3 (residues 3-187), and presumably is destroyed by its cleavage at Met 92; the Th cells that it induces are suppressible by each of the Ts-inducing peptides. In the GZ system, where the native antigen is quite large, the interactions between Th and Ts are highly circumscribed. This may be attributable to the topology of antigen fragments produced during processing; any relevant fragment must bear at least a Ts- and Th-reactive determinant to permit intercellular regulation. A final implication of these results is that, not only does the existence of a Th-inducing determinant depend on its being an appropriate distance from a B cell epitope, but the existence of Ts-inducing determinants likewise depends on the existence of a neighboring Th-B cell association.

Animals↗
PubMed1982

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. I. Helper cells have a more restricted specificity repertoire than proliferative cells.

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.

Animals↗