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S Kozlowski

Publications and source records attributed to S Kozlowski.

At least 19 recordsLinked to original sources
PubMed1995

CD27-CD70 interactions regulate B-cell activation by T cells.

CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis.

Antigens, CD↗
PubMed1995

Molecular analysis of the same HIV peptide functionally binding to both a class I and a class II MHC molecule.

Although several peptides have been found to bind to both class I and class II molecules, the basis for this binding of the same peptide to two classes of MHC molecules has not been compared previously. We have analyzed one such peptide, P18 from the V3 loop of HIV-1 gp160, which we have previously shown to be recognized by CD8+ CTL with the class I molecule H-2Dd, and by CD4+ Th cells with the class II molecule I-Ad. With the use of truncated and substituted peptides, we found that the minimal core peptides are very similar, that the residues required for class I binding precisely fit the recently identified consensus motif for peptides binding to Dd (XGPX[R/K/H]XXX(X) [L/I/F]), and that at least three of the same residues are involved in binding to class II I-Ad. In addition, several of the same residues are involved in TCR interaction when the peptide is presented by class I and class II molecules. Modeling shows results to be consistent with the crystal structure of a peptide-class II MHC complex. Thus, the recognition of this versatile peptide by CD4+ Th cells with class II MHC molecules and by CD8+ cytotoxic T cells with class I MHC molecules is remarkably similar in both the core peptide used and the role of different residues in the ternary complex.

Amino Acid Sequence↗
PubMed1994

Functional cell surface expression by a recombinant single-chain class I major histocompatibility complex molecule with a cis-active beta 2-microglobulin domain.

As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2Dd class I MHC molecule (SC beta Ddm) which has beta 2-microglobulin (beta 2m) covalently linked to the amino terminus of a full-length H-2Dd heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2Dd-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SC beta Ddm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27 degrees or 37 degrees C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SC beta Ddm can be stably expressed in beta 2m-negative cells, showing that the single-chain molecule uses its own beta 2m domain to achieve correct folding and surface expression. Furthermore, the beta 2m domain of SC beta Ddm, unlike transfected free beta 2m, does not rescue surface expression of endogenous class I MHC in the beta 2m-negative cells. This strict cis activity of the beta 2m domain of SC beta Ddm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.

Animals↗
PubMed1994

Minimal requirements for peptide mediated activation of CD8+ CTL.

A physical chemical model of T cell stimulation by class I-peptide complexes was developed and used to analyse in vitro studies of gamma-interferon release as a function of the number of peptide and MHC molecules. The analysis provided reasonable estimates of well identified parameters, including equilibrium constants and the minimum number of T cell receptor-class I-peptide ternary complexes on a presenting cell required to activate T cells. The latter number was estimated as 3-5 per T cell. This is in distinct contrast to estimates in the literature of the number of peptide-MHC complexes required for activity, which is necessarily larger. The analysis also predicted that activity is potentiated by interaction between class I molecules, even if one member of the pair is not bound by antigen. The analytical approach used in this paper may be applicable to other activation systems.

3T3 Cells↗
PubMed1993

Multiple pathways are involved in the extracellular processing of MHC class I-restricted peptides.

T cell stimulation by certain class I-restricted antigenic peptides, such as the HIV 1 gp160-derived peptide, P18, requires peptide processing by angiotensin-1 converting enzyme (ACE) in FCS. We observed that longer versions of P18 and the murine cytomegalovirus pp89-derived core peptide, pMCMV, which could stimulate T cell hybridomas in FCS, were not as sensitive to the ACE inhibitor captopril as P18. Using cell-free soluble murine class I MHC molecules and protease inhibitors, we found that there are pathways of differing efficiency that use enzymes other than ACE for the proteolytic processing of peptides in serum. The kinetics of the generation of T cell stimulatory activity among P18 variant peptides in serum differed with peptide length, and with the nature of amino and COOH-terminal extensions. Such processing occurs in human plasma as well as in FCS. The understanding of this processing, its kinetics, and its inhibitors can lead to better design of peptide-based therapies, including vaccines.

Amino Acid Sequence↗
PubMed1993

A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.

Genetically engineered or chemically purified soluble monovalent major histocompatibility complex (MHC) molecules, which have previously been used to study T cells, have not blocked cytotoxic T-cell responses. Here we describe a genetically engineered divalent class I MHC molecule which inhibits lysis of target cells by alloreactive cytotoxic T cells. This protein, H-2Kb/IgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, H-2Kb, and an immunoglobulin heavy chain polypeptide. The chimeric protein has serological and biochemical characteristics of both the MHC and IgG polypeptides. Nanomolar concentrations of H-2Kb/IgG inhibited lysis of H-2Kb-expressing target cells not only by alloreactive H-2Kb-specific T-cell clones but also by alloreactive H-2Kb-specific primary T-cell cultures. A direct binding assay showed high-affinity binding between the H-2Kb/IgG molecule and an H-2Kb-specific alloreactive T-cell clone. Unlabeled H-2Kb/IgG displaced 125I-labeled H-2Kb/IgG from T cells with an IC50 of 1.2 nM.

Amino Acid Sequence↗
PubMed1993

Role of conserved regions of class I MHC molecules in the activation of CD8+ cytotoxic T lymphocytes by peptide and purified cell-free class I molecules.

To analyze the molecular interactions involved in CD8+ cytotoxic T lymphocyte (CTL) recognition quantitatively, we developed a cell-free antigen presenting system. Genetically engineered soluble H-2Dd molecules coated on plastic microtiter plates could present HIV envelope peptide to an antigen-specific CTL clone, inducing it to produce IFN-gamma in the absence of accessory cells and their accessory or co-stimulatory molecules. The peptide-MHC complexes were functionally stable for over 24 h. The magnitude of T cell activation was dependent on the concentrations of both class I MHC molecule and the peptide, but was more sensitive to the concentration of the MHC molecule than to that of peptide. This result suggests that one MHC molecule can play more than one role in activating the CTL. One such role is the interaction between CD8 and a conserved region of class I MHC, as suggested by the finding that holding the total MHC concentration constant with an irrelevant class I MHC molecule (H-2Kb engineered to have the same alpha 3 domain as H-2Dd) made the T cell response less sensitive to the change in concentration of the relevant MHC molecule (H-2Dd). The irrelevant class I MHC molecule (H-2Kb), unable to present this peptide by itself, augmented the T cell response at lower concentrations of peptide. These results suggest that the conserved alpha 3 domain of the class I MHC heavy chain as well as polymorphic regions play an important role in T cell activation and that T cell interaction with MHC molecules not presenting peptide can still augment the response.

Amino Acid Sequence↗
PubMed1992

Endogenous peptides of a soluble major histocompatibility complex class I molecule, H-2Lds: sequence motif, quantitative binding, and molecular modeling of the complex.

To gain insight into the rules that govern the binding of endogenous and viral peptides to a given major histocompatibility complex (MHC) class I molecule, we characterized the amino acid sequences of a set of self peptides bound by a soluble analogue of murine H-2Ld, H-2Lds. We tested corresponding synthetic peptides quantitatively for binding in several different assays, and built three-dimensional computer models of eight peptide/H-2Lds complexes, based on the crystallographic structure of the human HLA-B27/peptide complex. Comparison of primary and tertiary structures of bound self and antigenic peptides revealed that residues 2 and 9 were not only restricted in sequence and tolerant of conservative substitutions, but were spatially constrained in the three-dimensional models. The degree of sequence variability of specific residues in MHC-restricted peptides reflected the lack of structural constraint on those amino acids. Thus, amino acid residues that define a peptide motif represent side chains required or preferred for a close fit with the MHC class I heavy chain.

Amino Acid Sequence↗
PubMed1992

A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity.

Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines.

Animals↗
PubMed1992

Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression.

We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.

Amino Acid Sequence↗
PubMed1992

Serum angiotensin-1 converting enzyme activity processes a human immunodeficiency virus 1 gp160 peptide for presentation by major histocompatibility complex class I molecules.

T cell stimulation by the human immunodeficiency virus 1 gp160-derived peptide p18 presented by H-2Dd class I major histocompatibility complex molecules in a cell-free system was found to require proteolytic cleavage. This extracellular processing was mediated by peptidases present in fetal calf serum. In vitro processing of p18 resulted in a distinct reverse phase high performance liquid chromatography profile, from which a biologically active product was isolated and sequenced. This peptide processing can be specifically blocked by the angiotensin-1 converting enzyme (ACE) inhibitor captopril, and can occur by exposing p18 to purified ACE. The ability of naturally occurring extracellular proteases to convert inactive peptides to T cell antigens has important implications for understanding cytotoxic T lymphocyte responses in vivo, and for rational peptide vaccine design.

Amino Acid Sequence↗
PubMed1992

Solution binding of an antigenic peptide to a major histocompatibility complex class I molecule and the role of beta 2-microglobulin.

The major histocompatibility complex-encoded class I molecule, a noncovalent dimer of a polymorphic 45-kDa heavy chain and a nonpolymorphic 12-kDa beta 2-microglobulin (beta 2m) light chain, binds peptide antigen prior to its interaction with T-cell antigen receptors. We report here that the binding in aqueous solution at 37 degrees C of a soluble purified murine major histocompatibility complex class I protein, H-2Lds (a soluble analogue of H-2Ld consisting of the alpha 1 and alpha 2 domains of H-2Ld, the alpha 3 domain and the C terminus of Q10b), to an antigenic peptide is controlled by the light-chain subunit beta 2m. Analysis of the equilibrium binding data favors a model in which two classes of peptide binding sites exist, the high-affinity class having an equilibrium constant for dissociation, KH, of 3.7 x 10(-7) M and accounting for 12% of the theoretically available sites. Studies of binding in the presence of excess beta 2m indicate that this increases the concentration of available high-affinity sites. These data are consistent with a ternary model in which high-affinity sites are generated by the interaction of beta 2m with the peptide-binding class I heavy chain.

Amino Acid Sequence↗
PubMed1992

Exercise performance, core temperature, and metabolism after prolonged restricted activity and retraining in dogs.

To study physiological effects of restricted activity (RA) and subsequent retraining, 10 male mongrel dogs (1-5 years) performed a submaximal exercise endurance test on a treadmill (12 degrees slope, 1.6 m.s-1) during kennel control, after 8 weeks of cage (40 cm-w x 80 cm-h x 110 cm-l) confinement, and after 8 weeks of retraining using the same treadmill protocol 1 h/d for 6 d/week. Compared with control endurance (172 +/- 19 min), endurance decreased to 102 +/- 15 min (delta = -41%, p less than 0.05) after RA and increased to 223 +/- 24 min (delta = +30%, p less than 0.05) after training: the respective final levels and changes in rectal temperature were 41.25 and +2.15 degrees C, 41.60 and +2.70 degrees C (NS), and 41.35 and +2.40 degrees C (NS), respectively. Resting and post-exercise blood glucose and lactate concentrations were unchanged in the three experiments. After RA, resting muscle glycogen was reduced from a control level of 49.9 +/- 4.3 to 34.1 +/- 4.5 mmol.kg-1 (delta = 32%, p less than 0.05) which returned to the control level of 58.4 +/- 3.5 mmol.kg-1 after retraining. Resting plasma FFA levels were unchanged, but the RA post-exercise change was decreased from a control level of +0.400 +/- 0.099 to +0.226 +/- 0.039 mmol.L-1 (p less than 0.05). Neither restricted activity nor training affected glucose tolerance significantly. The results indicated that RA reduces exercise endurance, the effectiveness of exercise thermoregulation, muscle glycogen stores, and the lipolytic response to exercise and to noradrenaline stimulation. All these changes were reversed following 8 weeks of retraining.

Animals↗
PubMed1991

Excess beta 2 microglobulin promoting functional peptide association with purified soluble class I MHC molecules.

T lymphocytes expressing alpha beta receptors recognize antigenic peptide fragments bound to major histocompatibility complex class I or class II molecules present on the surface membranes of other cells. Peptide fragments are present in the two available HLA crystal structures and recent data indicate that peptide is required for the stable folding of the class I heavy chain and maintenance of its association with the class I light chain, beta 2-microglobulin (beta 2m), at physiological temperature. To explain how the exogenous peptide used to create targets for cytotoxic cells bearing CD8 antigen could associate with apparently peptide-filled extracellular class I molecules, we hypothesized that stable binding of exogenous peptide to mature class I molecules reflects either the replacement of previously bound peptide during the well documented beta 2m exchange process or the loading of 'empty' class I heavy chains dependent on the availability of excess beta 2m. In either case, free beta 2m should enhance peptide/class I binding. Using either isolated soluble class I molecules or living cells, we show here that free purified beta 2m markedly augments the generation of antigenic complexes capable of T-cell stimulation.

Cell-Free System↗
PubMed1989

Effect of endurance training on cardiovascular response to static exercise performed with untrained muscles.

Eighteen male volunteers (20-23 years) were submitted to 13 weeks of training consisting of 30 min of exercise (at 50%-75% VO2max) on a bicycle ergometer, performed three times a week. Every 4 weeks the heart rate (HR), blood pressure (BP), and systolic time interval (STI) responses to the static handgrip (at 30% MVC) were examined. Before and after 13 weeks of training echocardiograms were recorded in seven subjects at rest and during the handgrip. Significant decreases in HR and BP responses to static exercise were found already after 4 weeks of training. Resting STI and left ventricular dimensions were not affected by training, but during the static exercise the pre-ejection period (PEP) and isovolumic contraction time interval (ICT) at the corresponding HR were significantly shortened after 8 weeks of training. The values of echocardiographic indices of left ventricular function obtained during exercise after training did not differ significantly from those found before training. It is concluded that endurance training of moderate intensity improves cardiac function during static exercise performed with untrained muscles.

Adult↗
PubMed1985

Plasma testosterone and catecholamine responses to physical exercise of different intensities in men.

Plasma testosterone, noradrenaline, and adrenaline concentrations during three bicycle ergometer tests of the same total work output (2160 J X kg-1) but different intensity and duration were measured in healthy male subjects. Tests A and B consisted of three consecutive exercise bouts, lasting 6 min each, of either increasing (1.5, 2.0, 2.5 W X kg-1) or constant (2.0, 2.0, 2.0 W X kg-1) work loads, respectively. In test C the subjects performed two exercise bouts each lasting 4.5 min, with work loads of 4.0 W X kg-1. All the exercise bouts were separated by 1-min periods of rest. Exercise B of constant low intensity resulted only in a small increase in plasma noradrenaline concentration. Exercise A of graded intensity caused an increase in both catecholamine levels, whereas, during the most intensive exercise C, significant elevations in plasma noradrenaline, adrenaline and testosterone concentrations occurred. A significant positive correlation was obtained between the mean value of plasma testosterone and that of adrenaline as well as noradrenaline during exercise. It is concluded that both plasma testosterone and catecholamine responses to physical effort depend more on work intensity than on work duration or total work output.

Adult↗
PubMed1984

Endocrine response to physical efforts with equivalent total work loads but different intensities in man.

The purpose of this study was to determine relationships between some exercise-induced neurohormonal changes and either exercise intensity, or its duration, or the total work output. Plasma catecholamine, cortisol, growth hormone and insulin concentrations were measured in healthy male subjects having performed three bicycle-ergometer exercise tests of the same total work output (2160 J kg-1) but differing in intensity and duration. The results showed that during the relatively short exercise plasma noradrenaline, adrenaline and cortisol responses depend more on work intensity than on the total work output. No relationship was demonstrated between the exercise-induced increases in plasma growth hormone concentration and work intensity or its duration, while the plasma insulin concentration was more decreased during the effort of lower intensity, but longer duration than during a short exercise of high intensity.

Adult↗