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Biomedical subjects

D M Ojcius

Publications and source records attributed to D M Ojcius.

At least 19 recordsLinked to original sources
PubMed1996

Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism.

Interleukin-10 (IL-10) is a recently described pleiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-10 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-10 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8+ and CD4+ T cells and macrophages at the sites of tumor growth, but abundant NK cells were localized at these sites. This unexpected finding was confirmed by showing that IL-10 inhibits most B16-F10 and Lox metastases in mice deficient in T or B cells (SCID and nu/nu mice), but not in those deficient in NK cells (beige mice or NK cell-depleted mice). However, IL-10 downregulation of pro-inflammatory cytokine production and/or recruitment of additional effector cells may also be involved in the anti-tumor effect at higher local concentrations of IL-10, since transfected B16 tumor cells expressing high amounts of IL-10 were rejected by normal, nu/nu, or SCID mice at the primary tumor stage, and there was still a 33% inhibition of tumor metastasis in beige mice.

Animals↗
PubMed1996

High incidence of Coxiella burnetii markers in a rural population in France.

Since Coxiella burnetii, the causative agent of Q fever, is often transmitted from goats and sheep to humans through aerosols, we examined the sera from 168 persons involved in goat breeding in the Centre region of France and 40 members of veterinary and medical staff from the same region for the presence of antibodies against C. burnetii. An immunofluorescence assay was used to detect the presence of antibodies of the IgG isotope against epitopes from phase II of C. burnetii, which are the first antibodies to appear in infected people, and from phase I, which reflect more chronic stages of the infection. Our serological survey showed that most of the tested sera were positive for C. burnetii markers, indicating at least an encounter with the bacterium. In the overall population of 208 subjects, 71% of the sera had antibodies against phase II epitopes (titres > or = 1:40). Among the goat farmers and their immediate families, 78% had antibodies against phase II and 33% against phase I (titres > or = 1:40). Considering only high titres (> or = 1:320), though, only 37% of the farmers had antibodies against phase II and 15% against phase I. Only 3 out of 12 veterinarians working in the field had high titres of antibodies against phase II and phase I, while none of 28 members of veterinary and medical laboratories had significant levels of antibodies. These results emphasize the need for closer surveillance of populations at risk for Q fever, to prevent the infection by C. burnetii from reaching chronic stages of the disease.

Agricultural Workers' Diseases↗
PubMed1996

Presentation of antigens derived from microorganisms residing in host-cell vacuoles.

Antigens presented by major histocompatibility complex molecules have been classified into those presented by 'endogenous' and 'exogenous' pathways. Some microorganisms reside within host-cell vacuoles that appear to avoid both pathways. Novel presentation mechanisms are being unraveled for these microorganisms, and their antigens, rather than being just peptides, can also consist of lipids or DNA fragments.

Animals↗
PubMed1995

pH and calcium dependence of hemolysis due to Rickettsia prowazekii: comparison with phospholipase activity.

Rickettsia prowazekii invades nucleated cells through phagocytosis and subsequently proliferates in the cytoplasm of the host cell. Hemolysis and a phospholipase A2 (PLA2) activity at neutral pHs have previously been reported; even though the phagosomal environment is most likely acidic. We here show that R. prowazekii and R. typhi also lyse erythrocytes at mildly acidic pHs, compatible with an early phagosomal compartment. For R. prowazekii, hemolysis at an acidic pH but not a neutral pH is enhanced by Ca2+, raising the possibility that more than one membranolytic factor may be produced by the rickettsiae. The rickettsiae alone display PLA2 activity, implying that the enzyme is of bacterial rather than erythrocyte or host cell origin. Moreover, the PLA2 activity requires divalent cations (Ca2+ or Mg2+), and, as with many extracellular PLA2s from other species, it has a preference for acidic over neutral phospholipids. The pH dependence of PLA2 is similar to that of the hemolysis without Ca2+, but in the presence of the hemolysis buffers (which contain Mg2+), there is no calcium-induced enhancement at acidic pHs. Thus, these rickettsiae are endowed with a membranolytic activity that could contribute to the escape of the bacteria from early phagosomal compartments, and it is likely that multiple toxins may be used for membrane lysis.

Animals↗
PubMed1994

Role for MHC class I molecules in selecting and protecting high affinity peptides in the presence of proteases.

Ag fragments derived from the cytosol are transported into the endoplasmic reticulum (ER) lumen, where they bind to nascent MHC class I molecules. However, it is not known whether only high affinity peptides enter the ER, or whether ER proteases must trim longer precursor peptides down to optimal size. To evaluate the feasibility of proteolytic fine trimming in vitro, soluble Kd and Kb were preincubated with peptides that bind to Kd or Kb and the mixture was exposed to three different proteases. Class I protected allele-specific peptides against proteolysis, whereas the other peptides were degraded to the amino acid level. When a Kd/S11E (SYIPSAEYILE) complex was immunoprecipitated after incubation with carboxypeptidase, both S11E and the optimal sized S9I (SYIPSAEYI) were found to be specifically bound to Kd. However, only S91 was recovered if S11E, Kd and carboxypeptidase were mixed at the same time, and there was no fine-trimming of bound S11E if high protease concentrations and short proteolysis times were used, which suggests that trimming occurs only in the unbound state. The amount of peptide that immunoprecipitated with Kd increased after carboxypeptidase treatment of Kd/S11E, implying that the peptide affinity had increased. Kd also protected S9I against proteolysis by a lysed microsome preparation, demonstrating that class I could also protect high affinity peptides in vivo. These results suggest that class I participates in the selection of high affinity peptides in the ER, by sampling transported unbound peptides are degraded by ER proteases or efflux back to the cytosol.

Amino Acid Sequence↗
PubMed1994

Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation.

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution, insect metamorphosis, the development of the immune and nervous systems, and embryogenesis. The typical nuclear changes taking place in apoptotic cells include extensive condensation of chromatin and internucleosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmentation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nuclei in an attempt to reconstitute in vitro the digestion of genomic DNA associated with apoptosis. Micrococcal nuclease was found to induce a typical pattern of DNA fragmentation, but did not give rise to chromatin condensation, whereas Ca2+/Mg2+ induced both chromatin condensation and DNA fragmentation in isolated mouse liver nuclei. When the endonuclease inhibitor ZnCl2 was used, the DNA fragmentation induced by Ca2+/Mg2+ in nuclei could be completely inhibited, but chromatin condensation still occurred. For comparison, intact liver cells were treated with valinomycin, a potassium ionophore, which gave rise to an atypical cell death, with chromatin condensation appearing without DNA fragmentation. Our results suggest that endonuclease activation in apoptosis is neither necessary nor sufficient to induce chromatin condensation, and that DNA fragmentation and chromatin condensation may be triggered through separate pathways during apoptosis.

Animals↗
PubMed1994

Ly-49-independent inhibition of natural killer cell-mediated cytotoxicity by a soluble major histocompatibility complex class I molecule.

Natural killer (NK) cells lyse their targets in a non-major histocompatibility complex (MHC)-restricted manner, and the cytotoxicity can be inhibited by a number of MHC class I allele products, suggesting that NK cells may have a "positive receptor" that recognizes the target and a "negative receptor" that receives an inhibitory signal from class I. Since negative receptors could also exert their effect by masking a positive ligand, we have determined whether there may be a direct interaction between class I and an NK surface receptor by measuring cytotoxicity in the presence of a soluble class I molecule, Kd. Soluble Kd at micromolar concentrations could efficiently block NK cell cytotoxicity, suggesting that class I has a direct effect on cytotoxicity, rather than masking another target cell ligand. Inhibition required that Kd be at least divalent, probably because of its higher affinity or its ability to cross-link the NK surface receptor. In addition, the effect was independent of the peptide used to load Kd, and there was inhibition of cytotoxicity of NK cells derived from either H-2d or H-2b mice. Finally, depletion of NK cells expressing Ly-49 had no effect on the specific inhibition by Kd, raising the possibility that NK cells are endowed with additional negative receptors besides Ly-49. Taken together, these results suggest that there may be a family of NK receptors recognizing different class I alleles, which can receive negative signals by directly binding to class I on the target cell surface.

Animals↗
PubMed1994

Picosecond fluorescence spectroscopy of a single-chain class I major histocompatibility complex encoded protein in its peptide loaded and unloaded states.

The tryptophan fluorescence properties of two different peptide complexes of the single-chain H-2Kd (SC-Kd) were studied by means of the single-photon counting technique. The latter enables time-resolved measurements of fluorescence intensity and anisotropy decay parameters relevant to structural and dynamic properties of proteins. While the isolated SC-Kd molecules in their 'original' purified form represent the unloaded state, i.e., containing endogenous low-affinity peptides, the loaded SC-Kd protein is obtained by introducing well-defined high-affinity peptides that replace the low-affinity ones. These two SC-Kd forms were found to exhibit different time-resolved tryptophan emission patterns; the unloaded complexes show a slightly faster fluorescence intensity decay rate than the loaded one. Three well-resolved time domains were distinguished in the anisotropy decay course of both forms: a short one in the picosecond range, an intermediate one of several nanoseconds, and a long one spanning several dozens to hundreds of nanoseconds. They are assigned to superposition contributions of (short- and long-distance) non-radiative energy transfer processes, to motions of the tryptophans, and to rotation of the whole protein globule. In the loaded SC-Kds, the first two processes were found to be attenuated. It is therefore suggested that upon binding of high-affinity peptides, the SC-Kd structure becomes more compact and certain tryptophans become less accessible to quenchers. The faster anisotropy decay observed in the unloaded form reflects both an enhancement in the energy-transfer between the tryptophans and an acceleration of their motions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Role of the CDR1 region of the TCR beta chain in the binding to purified MHC-peptide complex.

Single alanine substitutions were introduced into the CDR1 region of the beta chain of a Kd-restricted TCR. Mutants and wild-type TCR were attached to the zeta chain of the CD3 complex and expressed at the surface of a rat basophil cell line. Transfectants were tested for the binding of purified soluble Kd-peptide complexes. With this experimental system, accessory molecules are unlikely to play a major role and the contribution of each residue to the interaction can be addressed. Results show that all positions in the CDR1 region are involved in the binding to the Kd-peptide complex but at varying degrees. These effects are discussed in relation to a molecular model of the TCR. Comparison of these results with previous data obtained in a T cell hybridoma system suggests the existence of a threshold in the TCR affinity necessary for mature T cell activation.

Animals↗
PubMed1994

Is antigen processing guided by major histocompatibility complex molecules?

Major histocompatibility complex class I (MHC-1) molecules bind peptide fragments derived from cytosolic antigens, and class II (MHC-2) molecules bind fragments of proteins that enter the endocytic pathway. How peptides of the right affinity and size are generated in vivo is still the focus of intense research. Current data are consistent with the view that precursor peptides of varying length are produced in the cytosol and transported into the endoplasmic reticulum lumen where nascent MHC-1 could sample the peptides for their affinity. High-affinity peptides would form stable complexes with MHC-1, which are resistant to proteolysis by luminal enzymes; peptides unable to bind to MHC-1 presumably undergo proteolysis in the lumen. On the other hand, multiple mechanisms are probably used to load MHC-2. Some proteins denatured in the acidic and reducing environment of the endosomes most likely bind to MHC-2 through the antigen's immunodominant region, and the exposed portions of the antigen are degraded by endosomal proteases. Other antigens must first be proteolysed into peptide fragments, which compete among themselves for binding to MHC-2, whereas heat shock proteins could also contribute peptides for MHC-2 loading. Because of their respective loading modes, there is a partial correlation between the MHC-2 binding affinity of the protein fragments and their in vivo immunodominance, which may not necessarily be the case for MHC-1.

Antigen Presentation↗
PubMed1994

Cytolysis mediated by ionophores and pore-forming agents: role of intracellular calcium in apoptosis.

Apoptosis is a term used to describe certain forms of physiological cell death that occur during embryogenesis, differentiation, and normal cell turnover. Previous reports concerning the effects of calcium ionophores on rodent thymocytes and the pore-forming proteins perforin and staphylococcal alpha-toxin on murine tumor cells led to the suggestion that simply raising intracellular calcium causes apoptotic cell death. This hypothesis was tested using two ionophores, A23187 and valinomycin, and two pore-forming agents, melittin and staphylococcal alpha-toxin, on four murine tumor cell lines. Although treatment with these agents could raise intracellular calcium, and in some instances cause DNA fragmentation, only valinomycin caused apoptosis. In contrast to previous reports, our results suggest that raising intracellular calcium and inducing internucleosomal DNA fragmentation is not sufficient to elicit apoptotic cell death in all cell types.

Animals↗
PubMed1994

Major histocompatibility complex class I molecules and resistance against intracellular pathogens.

The immune system employs a temporal hierarchy of effector mechanisms to combat infections by intracellular pathogens. The nonspecific response is independent of MHC and can be activated rapidly, while the specific response is slower, more specific, and requires major histocompatibility complex (MHC) molecules. MHC-dependent responses have been characterized extensively in vitro for antigens presented by polymorphic MHC class Ia and class II proteins and recognized by T lymphocytes carrying alpha/beta T-cell receptors (TcR). Growing indirect evidence has implicated monomorphic MHC class Ib proteins and gamma/delta T lymphocytes in defense against bacterial infections, but the biochemical and immunological behavior of class Ib proteins and gamma/delta TcR has not been well characterized, and most hypotheses involving these proteins have relied on data obtained with polymorphic MHC proteins and alpha/beta TcR. An overview of studies describing bacterial infections in vivo suggests that, in many cases, MHC class I-dependent effector cells may not be indispensable for effective immune responses, exerting instead a modulatory effect during the course of infection. Furthermore, many class Ib proteins have probably specialized to present stress antigens and conserved microbial antigens, which may be recognized by gamma/delta T cells through an interaction that is qualitatively very different from alpha/beta TcR binding to class I and class II proteins.

Animals↗
PubMed1994

Major contribution of the beta chain to the antigen specificity of a T cell receptor.

T cell receptors (TCR) recognize peptides presented by major histocompatibility complex (MHC) molecules on the surface of cells. Sequence homology between the variable regions of the T cell receptor and of antibodies suggests that similarly-folded domains participate in ligand binding in both cases. However, most current models assume that both TCR chains (alpha and beta) are required for specific binding, whereas the heavy chain alone can confer specificity on many antibodies. We have therefore constructed chimeric molecules with alpha and beta from two different TCR, one restricted by the class II MHC protein, Ek, and the other by the class I MHC, Kd. The beta chain alone was sufficient for specific recognition of a peptide, Cw3, bound to Kd, but the alpha chain contributed to the overall avidity. These results suggest that other TCR may recognize their ligands primarily through the beta chain.

Epitopes↗
PubMed1993

Dissociation of the peptide/MHC class I complex: pH dependence and effect of endogenous peptides on the activation energy.

Dansylated peptides were used to characterize the dissociation of peptides from a recombinant class I major histocompatibility complex protein. Dissociation of endogenous, low-affinity peptides from the class I molecule Kd had an activation energy of 6.78 +/- 0.64 kcal/mol in the 14 to 26 degrees C temperature range, but there was a break in the Arrhenius plot between 12 and 14 degrees C. Dissociation of a dansylated, high-affinity peptide had an activation energy of 20.24 +/- 1.69 kcal/mol, and there was similarly a break in the plot. Both direct interactions between peptide and the class I heavy chain and indirect effects of the peptide affinity on the extent of light chain association with heavy chain may contribute to the difference in activation energies, while the break in the Arrhenius plots implies a temperature-dependent conformational change. Dissociation was also slowest at neutral pH, but the peptide/class I complex dissociated rapidly at pH greater than 9 and less than 5, suggesting that endocytosed class I proteins would most likely lose their bound peptides at the acidic pH of endosomes.

Amino Acid Sequence↗
PubMed1993

Dissociation of the peptide-MHC class I complex limits the binding rate of exogenous peptide.

Soluble, single-chain molecules for two MHC class I alleles, H-2Kd and H-2Kb, were used to analyze the kinetics of antigenic peptide binding to MHC. After MHC preloading with radiolabeled or fluorescent peptides, the observed rate of MHC-peptide complex dissociation increased after addition of an excess of unlabeled competitor peptide. Although exogenous peptides conforming to the allele-specific motif were required for the enhanced complex dissociation to occur, the dissociation rate of the complex was independent of exogenous peptide concentration. Similarly, the association rate of exogenous peptides was independent of concentration, reflecting the presence of low affinity peptides in the binding sites of the recombinant MHC proteins; the sequences of these endogenous peptides conform to the consensus motif for the MHC allele studied. Finally, the association rate of exogenous peptide decreased when MHC molecules were preloaded with high affinity peptides, and the binding of labeled high affinity peptide to isolated recombinant MHC was faster than the subsequent dissociation observed in the presence of competitor peptide. Taken together, these results imply that the rate of exogenous peptide binding is limited by the dissociation rate of the previously bound peptides.

Amino Acid Sequence↗
PubMed1993

Binding of alanine-substituted peptides to the MHC class I protein, Kd.

Peptides eluted from the native MHC class I molecule, Kd, are generally nonamers that display a strong preference for Tyr in position 2. We investigated the molecular basis for this 'consensus motif' by synthesizing a virally derived peptide, NP 147-155, that is known to be presented by Kd on living cells, and peptide variants of NP 147-155 in which the amino acids in the different positions were sequentially replaced by Ala. All of the peptides bound to purified Kd molecules in vitro with high affinity, except for the peptide in which Tyr2 was replaced by Ala, for which the affinity for Kd decreased at least 100-fold. These results confirm the interpretation of the in vivo studies; namely, that Tyr2 is a critical anchor residue for binding to Kd.

Alanine↗