Association of tyrosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T lymphocytes.

Accumulating data show that the tyrosine protein kinase Zap-70 plays an essential role in T cell receptor-mediated signal transduction. However, the model of action, as well as the physiologically relevant substrates of Zap-70, have not been determined. We have attempted to identify a 120-kD tyrosine-phosphorylated protein (p120) that associates with Zap-70 in activated T lymphocytes. The results of our analyses showed that p120 is largely encoded by the c-cbl protooncogene. Furthermore, the association of Zap-70 with c-Cbl was shown to be induced by T cell receptor stimulation, implying that it required posttranslational modification of one or both of these products. FynT, but not Lck, also associated with c-Cbl in activated T cells. Finally, using a heterologous system, it was demonstrated that the ability of Zap-70 to cause tyrosine phosphorylation of p120c-cbl was dependent on Lck- or FynT-mediated signals. As c-Cbl can associate with several other signaling molecules, it may couple Zap-70 to downstream effectors during T cell activation.

Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn.

Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production.

Regulation of T-cell antigen receptor signalling by Syk tyrosine protein kinase.

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.

The CD4 associated tyrosine protein kinase p56lck is positively regulated through its site of autophosphorylation.

Antibody-mediated aggregation of the CD4 T-cell surface antigen activates bound p56lck molecules and can result in increased intracellular tyrosine protein phosphorylation. To evaluate the basis of the CD4 induced tyrosine phosphorylation signal, we have studied the ability of CD4 to regulate the function of p56lck when these two molecules are co-expressed in non-lymphoid cells. Our studies indicate that cross-linking of CD4 is capable of activation of p56lck in fibroblasts in a manner analogous to that previously reported for T-lymphocytes. They also demonstrate that replacement of the major site of autophosphorylation of p56lck (tyrosine 394) by a phenylalanine residue abolishes the ability to activate p56lck by CD4 cross-linking, implying that this residue is critical for the positive regulation of the Lck tyrosine kinase activity by CD4. Contrary to what we have previously reported for an antigen-dependent murine T-cell clone, as well as murine thymocytes, the CD4 induced activation of p56lck observed in fibroblasts does not result in marked changes in Lck tyrosine phosphorylation, suggesting that other lymphoid specific components may be required for these tyrosine phosphorylation changes.

Regulation of the enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck by the non-catalytic SH2 and SH3 domains.

The enzymatic activity of the lymphocyte-specific tyrosine protein kinase p56lck appears to be tightly regulated by phosphorylation of the conserved carboxy-terminal tyrosine residue 505. Indeed, substitution of this tyrosine residue by a non-phosphorylatable phenylalanine results in a constitutively activated version of p56lck that can transform rodent fibroblasts. In this report, we evaluate the functions of the conserved non-catalytic Src homology (SH) domains 2 and 3 of p56lck in the regulation of its enzymatic activity in NIH3T3 fibroblasts. We found that deletion of the SH2 or, to a lesser extent, the SH3 domain of p56lck resulted in an increase in the tyrosine protein kinase activity of wild-type Lck polypeptides. The SH2 domain (but not the SH3 domain) was also required for full oncogenic transformation by Lck molecules activated through removal of tyrosine 505. This effect did not appear to be the result of a diminution of the enhanced catalytic activity of F505 Lck polypeptides. However, it may relate to the findings that the SH2 domain can bind and possibly enhance phosphorylation of specific phosphotyrosine-containing proteins. Taken together, these observations imply roles for the non-catalytic SH2 and SH3 domains in the regulation of the catalytic activity of p56lck. They suggest that the enzymatic function of this Src-related polypeptide is physiologically repressed by processes dependent on the presence of the SH2 and SH3 sequences. Moreover, they indicate that the SH2 domain also plays a positive role in the function of activated p56lck molecules in NIH3T3 cells.

Association of CD45 with Lck and components of the Ras signalling pathway in pervanadate-treated mouse T-cell lines.

Pervanadate treatment of a mouse T-cell hybridoma cell line overexpressing an activated form of p56lck was shown to result in tyrosine phosphorylation of CD45. Immunoprecipitates prepared under mild lysis conditions using antibodies against CD45 contained a number of other proteins, including p56lck, that were not evident in the absence of pervanadate treatment or in T cells lacking activated Lck, implying that under these conditions, CD45 is present within complexes containing Lck and other proteins. Analyses involving deletion mutants of p56lck indicated that interactions with CD45 did not absolutely require the SH2 and SH3 regions of Lck. Three proteins of the Ras signalling pathway were also shown to associated with CD45: the GTPase-activating protein for Ras (rasGAP), the signalling protein Grb2, and, possibly via complex formation with Grb2, the guanine nucleotide exchange factor mammalian son of sevenless (mSOS). In addition, CD45 was also found in immunoprecipitates prepared from these cells using an antiserum which recognizes Vav. It is possible that rasGAP, Grb2 and Vav bind to phosphotyrosine residues on CD45 via SH2 domains, and such interactions may be specific as other SH2-containing proteins, including phospholipase C alpha (PLC gamma), the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). She and Syp/PTP1D were not detectably associated with CD45 under the same conditions. These data suggested that in addition to its role as a protein tyrosine phosphatase, CD45 may participate in T-cell activation by serving as a membrane docking site for components of the Ras signalling pathway.

Two distinct protein isoforms are encoded by ntk, a csk-related tyrosine protein kinase gene.

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory tyrosine protein kinases, which we have termed Ntk. Intriguingly, the mouse ntk cDNA sequences published by two independent groups differed by the presence or absence of a 136 nucleotide-insert near their 5' ends. In this report, we demonstrate that this 136 nucleotide-sequence likely corresponds to a complete exon in the ntk gene (termed exon 2), and that the two types of cDNAs/transcripts are produced by alternative splicing. Using ribonuclease protection assays, it was also established that brain and lymphoid organs, as well as most hemopoietic cells, predominantly expressed ntk transcripts lacking exon 2. In contrast, selected hemopoietic cell lines, such as the immature myeloid cell lines 32D cl3(G) and WEHI-3B, exclusively possessed exon 2-bearing RNAs. Interestingly, exon 2 introduced a novel in-frame upstream AUG in the ntk transcript, which is in the appropriate context for translation initiation. Evidence was obtained that this AUG is utilized in vivo, and that it extends the amino-terminal sequence of Ntk by 40 amino acids. Indeed, while exon 2-deficient ntk RNAs were translated into a 52 kilodalton (kDa) polypeptide (p52ntk), those bearing exon 2 produced a 56 kDa protein (p56ntk). Furthermore, p56ntk, but not p52ntk, was recognized by an antiserum directed against the novel amino-terminal sequence encoded by exon 2. Additional biochemical characterizations showed that p52ntk and p56ntk were localized to the cytoplasm, and that they partially accumulated in the detergent-insoluble cellular fraction. This last finding suggested that the Ntk proteins can associate with the cytoskeleton. Finally, through linkage analysis of two multilocus crosses, the ntk gene was mapped to Chromosome 10 in the mouse. Taken together, these data showed that ntk, a csk-related tyrosine protein kinase gene, encodes two protein isoforms expressed in distinct cell types. Moreover, they raised the possibility that Ntk may be involved in the regulation of Src-like enzymes in detergent-insoluble cellular compartments.

Preclinical evaluation of alpha-1-proteinase inhibitor. Pharmacokinetics and safety studies.

To assess the pharmacodynamics and safety of alpha-1-proteinase inhibitor (human) (A1PI) isolated from pooled human plasma, a series of animal studies was conducted. Using both unlabeled and 125I-labeled A1PI (highly purified), plasma residence time and tissue distribution were determined in rabbits. A catabolic half-life of 48.5 hours was obtained for the labeled material, which agreed well with the antigenic decay (35.5 hours), measured with a specific enzyme-linked immunosorbent assay, and the functional activity decay (38.1 hours), measured antigenically by the ability of resident human A1PI to complex with human neutrophil elastase. No unusual tissue distribution was observed at the first, 24th, or 168th hour of sacrifice. Cynomolgous monkeys received infusions of labeled A1PI and a catabolic half-life of 55.45 hours was obtained; infusion of unlabeled material yielded anticipated plasma recovery and a significant increment in A1PI in bronchial-alveolar lavage fluid, both antigenically and functionally determined. Safety studies assessing acute physiologic response and both acute and subacute toxicity presented no significant adverse effects. We conclude that A1PI (human) presents normal pharmacodynamics and safety and is therefore associated with a wide margin of safety for the intended clinical applications.

Efficacy of antithrombin III supplementation in animal models of fulminant Escherichia coli endotoxemia or bacteremia.

Plasma antithrombin III (ATIII) levels decrease early during gram-negative septicemia, and even a moderate decrease in this major inhibitor of the coagulation system is associated with serious disseminated intravascular coagulation (DIC). Herein the efficacy of high-dose (at least 250 units/kg) ATIII supplementation in animal models of Escherichia coli endotoxemia or bacteremia is reported. An endotoxemic rat model demonstrated that: (1) DIC occurs very early, before the appearance of deleterious cardiovascular abnormalities; (2) ATIII prophylaxis attenuates DIC, metabolic dysfunction, and organ damage; (3) ATIII prophylaxis increases permanent survival; (4) ATIII treatment one hour after endotoxin challenge attenuates DIC, metabolic dysfunction, and organ damage, although not as well as when given prophylactically, and survival is not increased. An endotoxemic sheep pulmonary dysfunction model demonstrated that: (1) ATIII prophylaxis prevents the typical decrease in arterial oxygen partial pressure; (2) ATIII prophylaxis combined with alpha-1-proteinase inhibitor significantly attenuates indices of pulmonary dysfunction. An E. coli bacteremic baboon model demonstrated that ATIII prophylaxis and treatment significantly attenuate indices of DIC and organ damage and prevent death in an otherwise completely lethal dose bacterial challenge. In conclusion, prophylactic treatment with high doses of ATIII may be efficacious in disease states of impending disseminated intravascular coagulation, such as primary or secondary gram-negative septicemia.

Antithrombin inactivation by neutrophil elastase requires heparin.

In certain thrombotic states, large declines in the levels of functional circulating antithrombin occur, which may reflect the highly active nature of the endothelial surface in suppressing excessive amounts of activated coagulation enzymes. Alternatively, we have recently observed an unexpected and paradoxical in vitro functioning of heparin that could result in the inactivation of antithrombin in pathologic conditions. Specifically, antithrombin was rendered nonfunctional as an inhibitor of clotting enzymes as a result of a limited, heparin-dependent cleavage by neutrophil elastase. This inactivation occurred only in the presence of the active anticoagulant heparin fraction, which suggested that the heparin-antithrombin complex was the substrate for elastase attack. Interestingly, neutrophil elastase was found to bind tightly to heparin and heparin-like materials. Neutrophil elastase has been previously linked to nonspecific proteinolysis occurring in inflammatory thrombotic reactions. This affinity of both antithrombin and elastase for heparin suggests a novel mechanism of potential specificity. An important component of this hypothesis is the localization of the elastase/antithrombin reaction away from the high circulating levels of elastase inhibitors. The proposed inactivation of antithrombin on the vascular surface would likely occur only in pathologic states associated with neutrophil sequestration and activation. Nevertheless, this mechanism could lead to a localized reversal of the nonthrombogenic nature of the endothelium and potentially lead to significant reductions of functional antithrombin in certain disease states.

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

Modulation of the cellular immune responses to T-cell-dependent and T cell-independent antigens in lambs with induced bovine viral diarrhea virus infection.

Functional interaction between lymphoid cells and lymphotropic viruses is particularly evident for bovine viral diarrhea virus (BVDV) in cattle and its closely related virus, the border disease virus (BVDV) in sheep. The most important aspect of acute or chronic phases of BVDV or BDV infection was the host's increased susceptibility to secondary bacterial or viral infection. To study the ability of BVDV to alter the development of the cellular immune responses to concomitant inoculation with T cell-dependent and T cell-independent antigens, lambs were inoculated twice with rabbit RBC and Escherichia coli lipopolysacharide (LPS) and then were infected with a cytopathic strain of BVDV at postinoculation day 3. Leukopenia characterized by lymphopenia developed after BVDV infection. Increased [3H]thymidine incorporation was observed in resting or lectin-stimulated blood mononuclear cells in the first weeks after inoculation in BVDV-infected lambs, but was followed by decreased [3H]thymidine incorporation after the second inoculation for up to 8 weeks after initial inoculation. In contrast, transient decrease of blastogenic responses, associated with toxic effect of LPS, was detected in inoculated noninfected lambs, but was followed by stimulation of cellular immune responses. Inoculated noninfected lambs had good in vitro cellular immune response to rabbit RBC and LPS antigens, whereas lymphocytes from BVDV-infected lambs could not mount lasting cellular immune responses to antigens or BVDV. Results suggest that BVDV infection in lambs modulates the ability of lymphocytes to respond to lectins or antigenic stimuli according to the time after infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Oncogenic activation of p59fyn tyrosine protein kinase by mutation of its carboxyl-terminal site of tyrosine phosphorylation, tyrosine 528.

As a result of alternative splicing, the Src-related tyrosine protein kinase p59fyn consists of two distinct isoforms termed FynB and FynT. Whereas the first product accumulates principally in brain, the second is expressed in hemopoietic cells, especially in T-lymphocytes. There is increasing evidence that the Fyn proteins are critical for normal functions of neuronal and lymphoid cells. To better understand the regulation of the catalytic function of p59fyn, we have tested the effects of mutating the major site of in vivo tyrosine phosphorylation, tyrosine 528, on the biological and biochemical properties of this enzyme. Our studies showed that a tyrosine 528-->phenylalanine (Y528F) mutation converted either Fyn isoform into a dominant oncoprotein, capable of full transformation of rodent fibroblasts. However, while both Y528F p59fynT and Y528F p59fynB were able to transform NIH 3T3 cells, activated FynT molecules were consistently more efficient at this process. It was also found that expression of wild-type p59fyn or kinase-defective Y528F Fyn molecules failed to provoke transformation of NIH 3T3 cells, implying that the transforming capabilities of Y528F Fyn relied on deregulated catalytic activity. Contrary to an earlier study (Cheng, S. H., Espino, P. C., Marshall, J., Harvey, R., Merrill, J., and Smith, A. E. (1991) J. Virol. 65, 170-179), these findings showed that mutation of the conserved carboxyl-terminal tyrosine residue markedly stimulated the catalytic function of p59fyn in vivo, implying that dephosphorylation of tyrosine 528 is sufficient to produce biologically relevant activation of the Fyn kinase. Moreover, our results provided further indication that the two Fyn isoforms possess distinct biochemical activities that may dictate functional differences in normal cell physiology.

Conserved cysteine residues are critical for the enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck.

We have evaluated the possibility that conserved cysteine residues are critical for the enzymatic function of p56lck. Through oligonucleotide-directed mutagenesis, 5 Lck residues (cysteines 217, 224, 378, 464, and 475) were individually mutated to alanines, and the effects of these substitutions were tested in various in vitro and in vivo assays. We found that mutation of either of 2 cysteines located in the carboxyl portion of the kinase domain (cysteines 464 and 475) abolished the catalytic function of Lck. In addition, it was noted that alteration of cysteine 475 resulted in a dramatic reduction of the half-life of p56lck. These cysteine residues are highly conserved throughout the tyrosine protein kinase family, suggesting that they may play important functions in catalysis and/or substrate recognition.

Plasma protein fraction. Product improvement studies.

The vasodilative effects associated with the use of sodium acetate have been reported in the literature. Preparations of plasma protein fraction (PPF) substantially free of this vasodilator were developed. This paper presents and discusses experimental work on the improved product.

Preclinical review of anti-tumor necrosis factor monoclonal antibodies.

OBJECTIVE: To review the preclinical evidence for the role of tumor necrosis factor (TNF) in the pathogenesis of septic shock and to assess the preclinical efficacy of anti-TNF therapies for this clinical problem. DATA SOURCES: The international English language literature from 1986 to the present formed the basis of this review. MEDLINE was used to identify pertinent in vitro and animal studies pertaining to the pathobiology of TNF and the use of anti-TNF therapies, with special emphasis on antibody approaches. STUDY SELECTION: Those studies that focused on the mechanisms of action of TNF, its role in the inflammatory cascade, and the potential uses of anti-TNF therapies were emphasized. Investigations that described animal and human results served as the primary database. DATA EXTRACTION: Animal studies were selected based on the relevance of the model to the pathogenesis of the human clinical sepsis syndrome. Where they provided supportive evidence, patient studies were selected on the basis of study design. DATA SYNTHESIS: The administration of anti-TNF antibodies in baboons, monkeys, and other species that were administered lethal doses of bacteria or endotoxin suggest that this approach may limit organ damage and decrease the mortality rate caused by the septic shock syndrome. Therapy with anti-TNF monoclonal antibodies is reviewed. CONCLUSIONS: Bacterial challenge induces the release of TNF (among other mediators), which exerts both physiologic and toxic effects that may ultimately lead to organ dysfunction and death. New anti-TNF therapies such as anti-TNF antibodies appear to attenuate the injurious effects of TNF and promote survival in otherwise lethal septic shock animal models, suggesting a similar benefit might be obtained in the treatment of human septic shock.