Biomedical subjects

C T Baldari

Publications and source records attributed to C T Baldari.

At least 19 recordsLinked to original sources

PubMed1998

Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T-cell antigen receptor-dependent gene activation.

The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Expression of the T-cell-specific tyrosine kinase Lck in normal B-1 cells and in chronic lymphocytic leukemia B cells.

Src family kinases play a key role in mitogenesis. The exquisitely tissue-specific distribution of different Src family members suggests that a fine tuning of their expression might be a key prerequisite for cell homeostasis. We tested B cells from patients affected by B-cell chronic lymphocytic leukemia (B-CLL) for expression of Src family kinases. The T-cell-specific tyrosine kinase Lck was found to be expressed at significant levels in CLL B-cells. This finding could be accounted for either by ectopic expression of Lck in B-CLL or by specific expression of this kinase in normal B-1 cells, which are believed to be the normal counterpart of CLL B cells. To answer this question B cells from different sources, characterized by a different size of the B-1 subpopulation, were tested for Lck expression. The results show that Lck expression is a feature of CD5(+), B-1 cells, suggesting a potential role for Lck in the self-renewal capacity of this B-cell subpopulation and supporting the notion that B-1 cells are the subset undergoing oncogenic transformation in B-CLL. Furthermore, we show that the CD5(-), B-2 subpopulation, while normally lacking Lck expression, acquires the capacity to express Lck ectopically upon transformation by EBV.

B-Lymphocyte Subsets↗

PubMed1998

NF-AT-luciferase reporter T cell lines as tools to screen immunosuppressive drugs.

The development of safer analogues of immunosuppressants such as cyclosporin A and FK506 is an important goal for a number of clinical applications ranging from transplantation to the treatment of autoimmune diseases. Here we show the generation and the characterization of Jurkat T cell lines stably transfected with a reporter construct containing the firefly luciferase gene under the control of NF-AT. These lines specifically respond in a cyclosporin A-sensitive manner to T cell antigen receptor-derived signals. Due to the high levels of luciferase activity expression fewer than 1000 cells are required for detection of luciferase. In addition, a simplified luciferase assay allows to reduce both the manipulations and the time required for the assay, making these lines potentially useful models for the automated screening of cyclosporin A and FK506 analogues.

Cyclosporine↗

PubMed1997

T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease.

Chronic antral gastritis following Helicobacter pylori (Hp) infection is characterized by a cellular inflammatory infiltrate whose cytokines may represent a host-dependent factor influencing the outcome of the infection. The pattern of cytokines produced by the immunologically active cells in the gastric antrum was analyzed at the mRNA level in antral biopsies from five Hp-infected patients with duodenal ulcer and three Hp-negative dyspeptic controls. T cell clones were generated from parallel antral biopsies of the same Hp-infected patients and assessed for reactivity to Hp Ags, cytokine profile, and effector functions. Antral biopsies from all Hp-infected patients showed IFN-gamma, TNF-alpha, and IL-12, but not IL-4, mRNA expression, whereas no cytokine mRNA signal was found in the mucosa of controls. A total of 24 out of the 163 CD4+ T cell clones (15%) derived from Hp-infected patients proliferated in response to a Hp lysate; 11 clones (46%) also reacted with Cag-A, 2 with Vac-A, and 1 with urease. Upon Ag stimulation, 20 out of the 24 Hp-reactive clones (83%) produced IFN-gamma, but not IL-4 or IL-5 (Th1-like), whereas 4 produced IFN-gamma, IL-4, and IL-5 (Th0-like). All Hp-specific clones secreted high levels of TNF-alpha. At low T:B cell ratio, Hp-specific clones expressed Ag-dependent helper function for B cell proliferation and Ig production, whereas at higher T:B cell ratios, 15 Th1 and 2 Th0 clones lysed Ag-pulsed autologous EBV-transformed B cells. Results provide evidence for Hp-specific Th1 effectors in the gastric antrum of Hp-infected patients, where they may play a role in the genesis of either peptic ulcer or Hp-associated gastric B cell lymphoma.

Adult↗

PubMed1997

Different cytokine profile and antigen-specificity repertoire in Helicobacter pylori-specific T cell clones from the antrum of chronic gastritis patients with or without peptic ulcer.

Helicobacter pylori (Hp) infection almost invariably results in chronic antral gastritis, but only a proportion of patients develop peptic ulcer. Some Hp strains may be more ulcerogenic than others, but some ulcerogenic mechanisms may also depend on the type of the host immune response. In this study, the antigen specificity and the cytokine profile of 53 Hp-specific CD4+ T cell clones derived from the antral mucosa of five patients with Hp-induced uncomplicated chronic gastritis (CG) were assessed and compared with those of 34 Hp-specific CD4+ T cell clones derived from six Hp-infected patients with chronic gastritis and peptic ulcer (CG-PU). The majority (28/34; 82%) of gastric Hp-specific T cell clones from CG-PU patients expressed the Th1 profile and 17 (all Th1) of the 34 clones were specific for cytotoxin-associated protein (CagA). In contrast, 34 (64%) of the 53 Hp-specific gastric T cell clones derived from CG patients were able to secrete both Th1 and Th2 cytokines (Th0 profile) and only 36% expressed a polarized Th1 profile. The majority (85%) of Hp-specific clones from CG patients recognized Hp antigens other than CagA, since 13/53 (25%) were specific for urease, 6 (11%) for VacA, 6 (11%) for HSP and 20 (38%) for other undefined Hp antigens. Results provide evidence that the type of T helper cell response against Hp may vary according to the antigen involved and suggest that a polarized Th1 response may play a role in the genesis of peptic ulcer, whereas a local Th0 response, including interleukin-4 production, may represent an individual host factor which contributes to lower the degree of gastric inflammation and prevent ulcer complication.

Adult↗

PubMed1997

Uncoupling of T-cell antigen receptor and downstream protein tyrosine kinases in common variable immunodeficiency.

Patients with common variable immunodeficiency (CVID) are heterogeneous in the clinical manifestations of the disease and the underlying mechanisms leading to the immunodeficiency. Although the overt defect is an impairment in B-cell function, there is increasing evidence of primary T-cell dysfunctions in a proportion of patients with CVID. We have analyzed T-cells from six CVID patients for activation of both early and late events in response to TCR triggering. The data showed that T-cells from three of six CVID patients were defective in the capacity to initiate the TCR/CD3 signaling pathway by activating intracellular tyrosine kinases, associated with impaired proliferative responses to TCR/CD3 triggering. Since both surface expression of the TCR/CD3 complex and intracellular expression of key tyrosine kinases such as p56lek and ZAP-70 were normal in these patients, our data suggest a defect in the earliest step of TCR signal transduction.

Adult↗

PubMed1997

Gene activating and proapoptotic potential are independent properties of different CD4 epitopes.

CD4 engagement triggers an early signaling cascade which initiates late events such as transcription factor activation. The outcome of CD4 engagement is T-cell commitment to alternative, dramatically different fates, such as activation and apoptosis. We have tested a panel of anti-CD4 mAbs specific for different CD4 epitopes, as well as HIV-1 gp120, for the capacity to activate crucial early events such as enhancement of p56(lck) kinase activity and Shc phosphorylation. The same CD4 epitopes were characterized for their capacity both to deliver a gene activating signal and to program T-cells to activation dependent death. No correlation could be found between capacity of specific CD4 epitopes to deliver a gene activating signal and capacity to prime T-cells to apoptosis, suggesting that gene activating and proapoptotic potential are independent functions of CD4 epitopes. Furthermore, while triggering of the calcium pathway appears critical in NF-AT activation, optimal p56(lck) activation and Shc phosphorylation might be required for initiation of the apoptotic pathway.

Antibodies, Monoclonal↗

PubMed1997

The genes encoding the antibacterial sex-specific peptides ceratotoxins are clustered in the genome of the medfly Ceratitis capitata.

Ceratotoxins are antibacterial peptides produced in the female reproductive accessory glands of the medfly Ceratitis capitata. Their expression is not affected by bacterial infection, but is enhanced after mating and is modulated by juvenile hormone. Three different peptides, named ceratotoxins A, B and C, have been previously purified from the female accessory gland secretion and their amino acid and cDNA sequences have been determined. We report here the complete nucleotide sequences of four genes encoding closely related ceratotoxin peptides. One of them encodes a novel peptide, which we named ceratotoxin D. Restriction and nucleotide sequence analysis indicate that these ceratotoxin genes are organized in a large cluster spanning more than 26 kilobases of DNA. All ceratotoxin genes are coordinately expressed. Ceratotoxin transcripts appear in 2-3 day old adult females, and they reach a maximum in 6-7 day old females. The presence of highly conserved motifs in the upstream regions of all the sequenced ceratotoxin genes suggests the presence of common regulatory elements for all ceratotoxins.

Amino Acid Sequence↗

PubMed1996

Generation of a monoclonal antibody to a defined portion of the Helicobacter pylori vacuolating cytotoxin by DNA immunization.

The capacity of mammalian cells to express proteins encoded by plasmid vectors injected as naked DNA has opened the possibility to induce an immune response against protein antigens by DNA vaccination. We have used DNA immunization to generate a strong antibody response to a defined portion of the Helicobacter pylori vacuolating cytotoxin. A high specific monoclonal antibody has been derived from the splenocytes of an immunized mouse. Our data show that DNA immunization offers the possibility to obtain monoclonal antibodies following a significant shortcut with respect to traditional immunization protocols.

Animals↗

PubMed1996

Molecular characterization of ceratotoxin C, a novel antibacterial female-specific peptide of the ceratotoxin family from the medfly Ceratitis capitata.

Ceratotoxins A and B are antibacterial peptides produced by the sexually mature females of Ceratitis capitata. The gene expression is restricted to the female reproductive accessory glands, and is not affected by bacterial infection, but is enhanced by mating. We report here the purification and the amino acid sequence of ceratotoxin C, a novel member of the ceratotoxin family, the cloning of its cDNA and the analysis of its expression. Ceratotoxin C is coordinately expressed with the other members of the ceratotoxin family. Its antibacterial activity is directed against both Gram-negative and Gram-positive bacterial strains but it is lower than that of ceratotoxin A. We demonstrate in the genome of C. capitata the presence of at least three ceratotoxin genes which express, in the female accessory glands, a set of peptides presumably involved in the protection of the genital tract during fertilization.

Amino Acid Sequence↗

PubMed1996

The aminoterminal phosphotyrosine binding domain of Shc associates with ZAP-70 and mediates TCR dependent gene activation.

T-cell antigen receptor stimulation results in recruitment to the zeta chain and phosphorylation both of the syk family protein tyrosine kinase ZAP-70 and of the Shc adaptor protein, which transduces activating signals to Ras. Both ZAP-70 and Ras are required for T-cell activation. We have investigated the functional link between these two molecules in TCR signaling. She was found to associate with ZAP-70 in response to TCR triggering. This association was dependent on the presence of the aminoterminal phosphotyrosine binding (PTB) domain of She. The analysis of She binding to a potential PTB domain binding site on ZAP-70 confirmed the interaction of the She PTB domain with ZAP-70 and identified the ZAP-70 phosphotyrosine residue involved in this interaction. To test the role of the She PTB domain in transducing TCR derived signals we measured the effects of the isolated She PTB domain on the activation of the T-cell specific transcription factor NF-AT. The isolated She PTB domain was designed to compete non productively with endogenous She for binding to up-stream tyrosine phosphorylated proteins and thus interfere with coupling to regulators of Ras activation. A significant inhibition of NF-AT activation by TCR triggering was observed, showing a functional involvement of She in TCR signaling through its PTB domain and suggesting an important role for She association with ZAP-70.

Amino Acid Sequence↗

PubMed1996

Cyclosporin A sensitivity of the HIV-1 long terminal repeat identifies distinct p56lck-dependent pathways activated by CD4 triggering.

The CD4 co-receptor interacts with nonpolymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells. This interaction results in the mobilization of a number of signaling mediators shared by the T cell receptor (TcR) signaling pathway and thus amplifies TcR-generated signals. We have investigated the outcome of CD4 engagement on the activation of both cellular transcription factors and the HIV-1 long terminal repeat (LTR). We show that CD4 triggering activates different pathways of HIV LTR activation which can be identified by their sensitivity to the immunosuppressant cyclosporin A. The response of the inducible cellular transcription factors involved in HIVLTR activation shows that both nuclear factor (NF)-kappa B and NF-AT mediate a cyclosporin A-sensitive response to CD4, while AP-1 is at least in part responsible for the cyclosporin A-insensitive response. Both pathways can, however, be blocked by a kinase-defective dominant negative p56lck mutant, supporting an essential role for p56lck kinase activity in CD4-dependent signal transduction. A functional analysis of different CD4 epitopes using either anti-CD4 mAb or HIV-1 gp120 reveals a common epitope-specific activation of both the LTR and of the transcription factors NF-kappa B and NF-AT.

Autoantibodies↗

PubMed1996

A motif within the T cell receptor alpha chain constant region connecting peptide domain controls antigen responsiveness.

Mutant alphabeta TCRs were generated by replacing domains of the alpha and beta chain constant regions with homologous domains from TCR delta and gamma chains, respectively. Chimeric TCRs in which the alpha chain contains TCR delta chain sequences within the connecting peptide domain are unresponsive to alloantigens and superantigens, and have defective interactions with the CD3/zeta complex. Although these antigen-unresponsive TCRs undergo zeta chain phosphorylation upon stimulation with superantigen, they do not generate a full signal capable of producing IL-2. Mutant TCRs acquire signaling activity with a combination of superantigen and calcium ionophore, indicating a defect in calcium-mediated signaling. Finally, a conserved motif, FETDxNLN, present in the alpha chain connecting peptide domain, is disrupted in all signaling-defective TCRs. This conserved alpha chain connecting peptide motif might mediate the transfer of signals from the alphabeta heterodimer to the CD3/zeta complex.

Amino Acid Sequence↗

PubMed1995

p56lck plays a key role in transducing apoptotic signals in T cells.

The CD4 receptor synergizes with the T-cell antigen receptor (TCR) in helper T-cell activation. However CD4 cross-linking in the absence of simultaneous TCR engagement leaves the cells primed to activation dependent apoptosis. To assess the role of the CD4 associated protein tyrosine kinase p56lck in CD4 priming to apoptosis we have constructed Jurkat T-cell lines stably transfected with a constitutively active form of p56lck. These cells were constitutively primed to undergo apoptosis upon TCR crosslinking with specific antibodies. In addition the Jurkat JCaM1 line, which is defective for p56lck expression, was resistant to TCR induced apoptosis. These data indicate that p56lck is required for T-cell apoptosis and that CD4 priming of T-cells for antigen dependent apoptosis is due to inappropriate or partial activation of the p56lck signal transduction pathway.

Antibodies, Monoclonal↗

PubMed1995

Signals from the IL-1 receptor homolog, Toll, can activate an immune response in a Drosophila hemocyte cell line.

The Toll gene encodes an interleukin 1 receptor-like protein that mediates dorsoventral polarity in the Drosophila embryo. The possible involvement of Toll or Toll-like proteins also in the Drosophila immune response was investigated by overexpressing Toll10B, a constitutively active mutant protein, in the Drosophila blood cell line mbn-2. Induction of the Cecropin A1 (CecA1) gene, coding for a bactericidal peptide, was used as an indicator for the immune response. Toll10B was found to increase CecA1 transcription, as detected with a cotransfected CecA1-lacZ reporter gene construct. This effect depends on the presence of a kappa B-like site in the CecA1 promoter. The endogenous Toll gene is expressed in mbn-2 cells, indicating that this gene may normally play a role in Drosophila blood cells.

Animals↗

PubMed1995

cDNA sequence and expression of the ceratotoxin gene encoding an antibacterial sex-specific peptide from the medfly Ceratitis capitata (diptera).

Ceratotoxins are antibacterial 3-kDa molecular mass amphiphilic peptides isolated from the female reproductive accessory glands of the medfly Ceratitis capitata. They are physiologically related to bee melittin and show amino acid sequence homology with magainin peptides. In this paper, we report the complete sequence of cDNA coding for ceratotoxin A and the expression of the gene during the life cycle of the insect. Experimental data show that the ceratotoxin is a gene expressed exclusively in the imaginal stages and that it is female-specific, related to sexual maturity, and stimulated by mating. Differently from most antibacterial insect hemolymph peptides, it is not induced by microbial infection. Western blot analysis using an anti-ceratotoxin antibody indicates the female accessory glands as the only site where the production of the ceratotoxin peptide occurs.

Amino Acid Sequence↗

PubMed1995

Inhibition of CD4/p56lck signaling by a dominant negative mutant of the Shc adaptor protein.

T-cell antigen receptor stimulation results in phosphorylation of the SH2 containing Shc proteins and recruitment of the Grb2/mSos complex suggesting that Shc proteins are involved in transducing T-cell activating signals to Ras. We have measured the effects of the isolated Shc-SH2 domain and the dominant negative RasN17 protein on activation of the T-cell specific transcription factor NF-AT. The isolated Shc-SH2 domain was designed to compete with endogenous Shc binding to upstream tyrosine phosphorylated proteins and to interfere with coupling to regulators of Ras activation. We have demonstrated that both the Shc-SH2 domain and the RasN17 protein significantly inhibited NF-AT activation by the CD4 coreceptor and the CD4 associated tyrosine kinase p56lck. In contrast, only the RasN17 protein reduced NF-AT activation by the TCR/CD3 complex. Furthermore, tyrosine kinase activity and p56lck protein were found in complexes immunoprecipitated with Shc specific antisera after CD4 triggering but not after CD3 triggering. These results indicate that both CD4 and CD3 signal to Ras and that this signaling is mediated by independent pathways of activation of the Shc adaptor protein.

Base Sequence↗

PubMed1995

Interactions between the tyrosine kinases p56lck, p59fyn and p50csk in CD4 signaling in T cells.

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.

CD4 Antigens↗