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T Lea

Publications and source records attributed to T Lea.

At least 19 recordsLinked to original sources
PubMed2003

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice.

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.

Amino Acid Sequence↗
PubMed2002

Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels.

Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed. The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

Blood Platelets↗
PubMed2000

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity.

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

cAMP-dependent protein kinase (PKA) inhibits T cell activation by phosphorylating ser-43 of raf-1 in the MAPK/ERK pathway.

cAMP-dependent protein kinase (PKA) has been suggested to interfere with T-cell activation by inhibiting interleukin (IL-2) receptor alpha-chain (CD25) expression and IL-2 production. The Ras/MAP kinase pathway has been found to be necessary for induction of the IL-2 production. In this study, we have scrutinized the Ras/MAP kinase pathway in Jurkat T-cells to attempt to identify any sites for PKA-mediated regulatory phosphorylations. Here we unambiguously demonstrate that PKA directly inhibits anti-CD3-induced MAP kinase activation. In vitro phosphorylation experiments showed that Raf-1 was extensively phosphorylated by PKA, while ERK2 and MEK were not. Phosphopeptide mapping identified Ser-43 of Raf-1 as the only site phosphorylated by PKA in the Ras/MAPK pathway. Transient transfection experiments demonstrated that mutations of Ser-43 of the Raf-1 kinase were rendered insensitive to cAMP-mediated inhibition.

Cyclic AMP↗
PubMed1999

Presence of vitronectin and activated complement factor C9 on ventriculoperitoneal shunts and temporary ventricular drainage catheters.

OBJECT: The pathogenesis of cerebrospinal fluid (CSF) shunt infection is characterized by staphylococcal adhesion to the polymeric surface of the shunt catheter. Proteins from the CSF--fibronectin, vitronectin, and fibrinogen--are adsorbed to the surface of the catheter immediately after insertion. These proteins can interfere with the biological systems of the host and mediate staphylococcal adhesion to the surface of the catheter. In the present study, the presence of fibronectin, vitronectin, and fibrinogen on CSF shunts and temporary ventricular drainage catheters is shown. The presence of fragments of fibrinogen is also examined. METHODS: The authors used the following methods: binding radiolabeled antibodies to the catheter surface, immunoblotting of catheter eluates, and scanning force microscopy of immunogold bound to the catheter surface. The immunoblot showed that vitronectin was adsorbed in its native form and that fibronectin was degraded into small fragments. Furthermore, the study demonstrated that the level of vitronectin in CSF increased in patients with an impaired CSF-blood barrier. To study complement activation, an antibody that recognizes the neoepitope of activated complement factor C9 was used. The presence of activated complement factor C9 was shown on both temporary catheters and shunts. CONCLUSIONS: Activation of complement close to the surface of an inserted catheter could contribute to the pathogenesis of CSF shunt infection.

Adsorption↗
PubMed1998

Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites.

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Vitronectin-binding staphylococci enhance surface-associated complement activation.

Coagulase-negative staphylococci are well recognized in medical device-associated infections. Complement activation is known to occur at the biomaterial surface, resulting in unspecific inflammation around the biomaterial. The human serum protein vitronectin (Vn), a potent inhibitor of complement activation by formation of an inactive terminal complement complex, adsorbs to biomaterial surfaces in contact with blood. In this report, we discuss the possibility that surface-immobilized Vn inhibits complement activation and the effect of Vn-binding staphylococci on complement activation on surfaces precoated with Vn. The extent of complement activation was measured with a rabbit anti-human C3c antibody and a mouse anti-human C9 antibody, raised against the neoepitope of C9. Our data show that Vn immobilized on a biomaterial surface retains its ability to inhibit complement activation. The additive complement activation-inhibitory effect of Vn on a heparinized surface is very small. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM131, complement activation on a surface precoated with Vn occurred as it did in the absence of Vn precoating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-precoated surface was significantly decreased. The results could be repeated on heparinized surfaces. These data suggest that Vn adsorbed to a biomaterial surface may serve to protect against surface-associated complement activation. Furthermore, Vn-binding staphylococcal cells may enhance surface-associated complement activation by blocking the inhibitory effect of preadsorbed Vn.

Animals↗
PubMed1996

Caffeine and micromolar Ca2+ concentrations can release Ca2+ from ryanodine-sensitive stores in crab and lobster striated muscle fibres

Ca2+ release mechanisms were studied in striated muscle from the walking legs of crabs using isometric tension recordings from isolated myofibrillar bundles. Caffeine-induced phasic contractions had properties consistent with Ca2+ release from a sarcoplasmic store, which could be optimally loaded in the presence of ATP at pCa 6.4­6.1. Ryanodine (10 µmol l-1) abolished the caffeine-induced contractions and in solutions with low Ca2+ buffering (0.1 mmol l-1 EGTA) itself caused phasic contractions, indicative of Ca2+ release. Ca2+-induced Ca2+ release (CICR) was observed in a pCa 5.8 solution (buffered by 1 mmol l-1 EGTA) as a phasic contraction of variable nature, inhibited by ryanodine (10 µmol l-1), procaine (10 mmol l-1) or benzocaine (5 mmol l-1). Ca2+ release was measured as a function of releasing pCa by using the force­time integral of the caffeine-induced contraction as an estimate of the Ca2+ remaining in the store. After the Ca2+ store had been loaded for 2 min at pCa 6.6, CICR was measured in the presence of 1 mmol l-1 Mg2+, 1 mmol l-1 EGTA and 5 mmol l-1 ATP. The threshold pCa for CICR was 6.0­6.4 under these conditions and more than 90 % of stored Ca2+ was released in 1 min by pCa values in the range 3.5­5.3. Benzocaine totally inhibited the release and promoted extra Ca2+ loading. Preliminary experiments showed a similar caffeine-releasable store in lobster abdominal muscle, which was slightly less sensitive to free [Ca2+]. It is concluded that in crustacean muscle caffeine and micromolar [Ca2+] can release Ca2+ from a ryanodine-sensitive store, which in many respects is similar to the sarcoplasmic reticulum of vertebrate skeletal and cardiac muscle.

Journal Article↗
PubMed1995

Effects of ochratoxin A upon early and late events in human T-cell proliferation.

We have investigated the effect of ochratoxin A (OA) on both early and late events during activation of human T lymphocytes. As early activation parameters we chose to measure both changes in intracellular Ca2+ levels and the activity of protein kinase C following the triggering process. Our results demonstrate that concentrations of OA that inhibit overall activation had no effect neither on the absolute levels nor on the duration of the Ca2+ response. Furthermore, PKC activity as measured by phosphorylation of two specific cytosolic substrate substrate proteins, was also unaffected. However, when the cells were prestimulated for 48 h to measure effects on late events in the activation cascade, addition of graded concentrations of ochratoxin A down to 6.4 microns completely inhibited the DNA synthesis. This shows that ochratoxin A is able to block DNA synthesis efficiently even if the activation process has been running for 48 h in advance. To investigate whether the inhibitory effect on DNA synthesis could be ascribed to inhibition of protein synthesis, we carried out experiments to measure protein synthesis both in resting and activated T-cells. In resting T-cells protein synthesis was nearly abrogated by 12.5 microM ochratoxin A, but only minor effects were observed in stimulated cells. The low impact of ochratoxin A on protein synthesis in activated cells may indicate that other mechanisms than just a general inhibition of protein synthesis are operating.

Calcium↗
PubMed1995

Freshly isolated tumour-infiltrating T-lymphocytes have a high cytotoxic potential, as measured by their ability to induce apoptosis in the target cell.

To test if freshly isolated tumour-infiltrating lymphocytes (TIL) can induce apoptosis in a target cell, we have combined two previously described methods. Because TIL predominantly are T-lymphocytes, we have applied a redirected approach. When the target cells that express anti-human-CD3 monoclonal antibodies in their membranes bind to the T cell receptor-associated CD3-complex, signals are generated, which activate T cell effector mechanisms. This approach circumvents problems with MHC-restriction and allows for functional testing of all T cells, irrespective of their clonal specificity. In order to assay for induction of DNA fragmentation, we have labelled the target cell nuclei with [3H]thymidine. Upon harvesting fragmented DNA are washed away. Electrophoretic analysis of the fragmented DNA demonstrated the characteristic 'ladder' pattern, consistent with apoptosis. This rapid and simple assay monitors the capacity of different T cells to induce apoptosis in the target cell. It depends on intercellular interactions and clearly discriminates between different T cell subsets. With this assay we demonstrate the functional integrity of the cytotoxic effector arm of freshly isolated TIL.

Adenocarcinoma↗
PubMed1995

Evidence for monoclonal expansion of synovial T cells bearing V alpha 2.1/V beta 5.5 gene segments and recognizing a synthetic peptide that shares homology with a number of putative autoantigens.

A peptide of 15 amino acids derived from the cereal glycine-rich cell wall protein (GRP), sharing a significant homology with Epstein-Barr virus nuclear antigen-1 (EBNA-1), fibrillar and procollagen, stimulated synovial fluid (SF) T cells from juvenile (JRA) and adult (RA) rheumatoid arthritis patients. An overexpression of the V alpha 2 gene family was found in the SF from patients who responded significantly to the peptide. To investigate in more detail the SF T-cell responses to the GRP peptide, we established peptide-specific T-cell lines and clones from a DR8+ positive JRA patient with pauciarticular form. The T-cell clones were phenotyped as T-cell receptor (TCR)alpha beta+/CD4+ and their clonality was investigated by polymerase chain reaction (PCR) and flow cytometric analysis. TCR sequences from different clones demonstrated that the clones were identical and used the V alpha 2.1/J alpha 6 combined with V beta 5.5/J beta 2.7 gene segments. Interestingly, direct sequencing of the V alpha 2 family PCR product obtained from cDNA prepared from freshly isolated SF mononuclear cells identified the same TCR sequence as that used by the clones, suggesting the monoclonality of SF CD4+ T cells bearing V alpha 2.1/J alpha 6 gene products. The present data suggest a recruitment and expansion of a SF T-cell subpopulation, and also support the hypothesis that autoimmune diseases can be triggered by protein epitopes with crucial amino acids homologous to self-proteins.

Amino Acid Sequence↗
PubMed1994

Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex.

Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase type I (cAKI), but not type II, is sufficient to mediate inhibition of T cell replication induced through the antigen-specific T cell receptor-CD3 (TCR-CD3) complex. Immunocytochemistry and immunoprecipitation studies of the molecular mechanism by which cAKI inhibits TCR-CD3-dependent T cell replication demonstrated that regulatory subunit I alpha, along with its associated kinase activity, translocated to and interacted with the TCR-CD3 complex during T cell activation and capping. Regulatory subunit II alpha did not. When stimulated by cAMP, the cAKI localized to the TCR-CD3 complex may release kinase activity that, through phosphorylation, might uncouple the TCR-CD3 complex from intracellular signaling systems.

Carrier Proteins↗
PubMed1994

Differential effects of cyclic adenosine 3',5'-monophosphate on T cell cytotoxicity.

We have investigated natural killer cell and T cell cytotoxicity using different assays and report a dual effect of cyclic adenosine 3',5'-monophosphate (cAMP) on T cell cytotoxicity depending on the activation status of the effector cell and the test system in question. cAMP enhanced the capacity of pre-activated T cells to induce DNA fragmentation in the target cell, while it inhibited spontaneous T cell cytotoxicity and natural killer cell cytotoxicity in conventional assays based on 51Cr release. The enhancement was most likely mediated by the cAMP-dependent protein kinase type II (cAKII), which is the particular isoform in T cells associated with the centrosome and the microtubule organizing center (MTOC). We show the complete co-localization of the cAKII with the centrosome after conjugate formation. Furthermore, the reorganization of the MTOC following conjugate formation brings the type II kinase into close proximity with the T lymphocyte membrane are engaged in the effector-target interaction. Functional studies utilizing different cAMP-analog combinations further substantiate the involvement of the type II kinase.

Cyclic AMP↗
PubMed1994

Limited heterogeneity in the T-cell receptor V-gene usage in lymphocytes infiltrating human colorectal tumours.

The presence of T lymphocytes in solid tumours may reflect an ongoing immune response against the transformed cells. We have used polymerase chain reaction (PCR) technology to investigate the T-cell receptor variable-region gene (V-gene) usage in freshly isolated tumour-infiltrating lymphocytes (TILs) to look for a possible oligoclonality of T cells in the tumour area. We used 19 different V beta-family-specific primers. Peripheral blood lymphocytes and lamina propria lymphocytes from the same patients were also tested by PCR. Our results demonstrate a limited heterogeneity in the V-gene usage of TILs from seven patients with colorectal cancers, suggesting a local antigen-driven immune response at the tumour site.

Adenocarcinoma↗
PubMed1994

Dietary supplementation with very long-chain n-3 fatty acids in man decreases expression of the interleukin-2 receptor (CD25) on mitogen-stimulated lymphocytes from patients with inflammatory skin diseases.

T-cell activation and cytokine production play an important role in several chronic inflammatory diseases. Because n-3 fatty acids exert beneficial effects on the clinical state of some of these diseases, we examined the effect of dietary supplementation of n-3 fatty acids on T-cell proliferation, expression of CD25 (interleukin-2 receptor alpha-chain), secretion of interleukin-2, interleukin-6 and tumour necrosis factor from T-cells from patients with psoriasis and atopic dermatitis. During 4 months, 21 patients supplied 6 g of highly concentrated ethyl esters of EPA and DHA in gelatin capsules daily to their diet. In the control group 20 patients supplied 6 g per day of corn oil in gelatin capsules to their diet. Eicosapentaenoic acid (20:5, n-3) of serum phospholipids increased from 14 (min 4-max 42) to 81 (min 59-max 144) mg l-1 (P < 0.01) in patients with atopic dermatitis receiving n-3 fatty acids, and from 25 (min 7-max 66) to 74 (min 46-max 142) mg l-1 (P < 0.01) in patients with psoriasis, whereas docosahexaenoic acid (22:6, n-3) increased from 65 (min 46-max 120) to 92 (min 54-max 121) mg l-1 (P < 0.05) and from 81 (min 38-max 122) to 92 (min 63-max 169) mg l-1 (NS) in atopic and psoriatic patients, respectively. The changes in the serum phospholipid fatty acid profile in the groups receiving n-3 fatty acids, correlate to the dietary intake of corresponding fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1994

Cyclic AMP sensitive signalling by the CD28 marker requires concomitant stimulation by the T-cell antigen receptor (TCR/CD3) complex.

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RI alpha 2-C beta 2) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anti-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP).

CD28 Antigens↗
PubMed1994

Human colorectal tumour infiltrating lymphocytes express activation markers and the CD45RO molecule, showing a primed population of lymphocytes in the tumour area.

This study investigated the phenotype of freshly isolated human tumour infiltrating lymphocytes (TIL) from 14 patients with colorectal tumours, and compared them with lymphocytes derived from the lamina propria of the unaffected mucosa and with lymphocytes derived from peripheral blood of the same patients. It was found that TIL expressed the activation markers CD25 and HLA-DR to a higher extent than the peripheral blood lymphocytes (p = 0.01), and that both lamina propria lymphocytes and TIL preferentially expressed the CD45RO + phenotype, associated with memory cells, in contrast with peripheral blood lymphocytes [corrected]. Both lamina propria lymphocytes and TIL contained few natural killer (NK) cells (CD3-CD56+) compared with peripheral blood lymphocytes (p = 0.001), and this was reflected in the cytotoxicity assays. After 1 to 2 weeks in culture with interleukin-2 100 U/ml, lymphocytes from all three compartments had a high cytolytic activity against all targets tested, consistent with the lymphokine activated killer cell phenomenon. No increase in the number of NK cells was noted after culture, but 20-30% of the T cells now coexpressed the CD56 molecule. This was most prominent in the CD8+ subset, but lymphokine activated killer cell activity was found in both CD4+ and CD8+ subsets. Possible tumour escape mechanisms are discussed.

Colonic Neoplasms↗