Fast bedside measurement of blood count and C-reactive protein in newborns compared with conventional methods.

BACKGROUND: Abnormal complete blood count (CBC) and high plasma C-reactive protein (CRP) are associated with neonatal infections and could be helpful in the diagnosis of neonatal sepsis and to monitor the antibiotic treatment. OBJECTIVES: The aim of this work is to evaluate and compare the performance of a bedside analyzer for blood count and C-reactive protein (CRP) with a conventional analyzer in a neonatal population. METHODS: 150 capillary or venous blood samples of term and preterm newborns were processed on an ABX-MicrosCRP200 analyzer and on a SysmexXE2100 (conventional hematology analyzer) for CBC, leukocyte differential, reticulocytes, and nucleated red blood cells (NRBC); high-sensitivity CRP (hs-CRP) was performed on a ModularPE. The differences between complete blood count and CRP were regressed against their means and assessed by means of intra-class-correlation. RESULTS: The intra-class-correlation for white blood cell (WBC) was 0.98, for hemoglobin 0.97, for hematocrit 0.96, for mean corpuscular volume 0.95, and for platelet 0.98. ABX-MicrosCRP200 overestimated the WBC (+1.27 x 10(3)/microL; p < 0.001), hematocrit (+1.80%; p < 0.001), and platelet (+13.55 x 10(3)/microL; p < 0.001). The intra-class-correlation for CRP was high (0.97), without systematic difference between the two values (p = 0.64). CONCLUSIONS: The agreement between the two methods was high for both tests. However, the SD of the difference for WBC and platelet could be clinically important in leukopenic or thrombocytopenic newborns.

Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts.

The Shc adaptor is responsible for coupling receptor tyrosine kinases and tyrosine kinase-associated receptors to the Ras/MAP kinase pathway. Shc is believed to be regulated by a change in subcellular localization from the cytosol to the plasma membrane, where it recruits Grb-2/Sos complexes and hence permits juxtaposition of the guanine nucleotide exchange factor Sos to Ras, resulting in GDP/GTP exchange and Ras activation. Shc has been recently shown to inducibly colocalize in detergent-resistant membrane rafts together with the activated TCR and associated signaling molecules. To understand whether Shc localization in membrane rafts is sufficient to regulate Shc function, we constructed a Shc chimera containing the Ras membrane localization motif at the C-terminus. We show that membrane targeted Shc was constitutively localized in the plasma membrane of T-cells, and was mostly compartmentalized in lipid rafts. Membrane targeted Shc was phosphorylated on tyrosine residues and bound Grb-2/Sos in the absence of TCR engagement. Furthermore, expression of membrane targeted Shc resulted in constitutive downstream signaling, including Erk2 activation and enhancement of TCR dependent activation of the TCR responsive transcription factor NF-AT. Hence localization of Shc in membrane rafts is sufficient for Shc to acquire a signaling competent state. Interestingly, a membrane targeted Shc mutant lacking both Grb-2 binding sites was not only incapable of signaling in the absence of TCR triggering, but transdominantly inhibited endogenous Shc, supporting a non redundant role for Shc in the activation of the Ras/MAP kinase pathway in T-cells.

Restoration of thymic development in an Lck(-/-) thymoma overexpressing ZAP-70.

Thymic development is strictly controlled by Src and Syk family protein tyrosine kinases. The major players in this process are Lck and ZAP-70, which regulate critical differentiation steps of thymopoiesis. Notwithstanding the critical role of Lck and ZAP-70 in thymocyte development as compared to the related kinases Fyn and Syk, a partial functional redundancy between members of the same family of protein tyrosine kinases has emerged from studies on genetically manipulated mouse models. Furthermore, a close functional interplay between Lck and ZAP-70 in intracellular signaling has been shown to occur in thymocytes. Here we present the characterization of a thymoma from an Lck(-/-) mouse, where the block in thymocyte development is overcome and the transition between the CD4(-)CD8(-) and CD4(+)CD8(+) stages is fully restored. Determination of the expression levels of Fyn, ZAP-70 and Syk in thymocytes form the Lck(-/-) thymoma revealed high levels of ZAP-70 overexpression and recovery of a specific subset of phosphoproteins as compared to Lck(-/-) thymocytes. Hence ZAP-70 overexpression in thymocytes is associated with recovery from the developmental arrest caused by the absence of Lck, suggesting a role for ZAP-70 downstream of Lck in the maturation of CD4(+)CD8(+) thymocytes.

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.

Dysregulation of the protein tyrosine kinase LCK in lymphoproliferative disorders and in other neoplasias.

Initially identified as a T-cell specific member of the Src family of protein tyrosine kinases, Lck has become the object of intensive investigations which have revealed a key role for this kinase in the central processes controlling T-cell development, activation, proliferation and survival. Experimental evidence of the oncogenic potential of Lck, together with the identification of defects in the regulation of Lck expression or activity in T-cell leukemias, suggests that dysregulation of Lck might play a role in neoplastic transformation. Here we review the data documenting a potential role for this kinase in the initiation and maintenance of the transformed state in human cancers.

A siderophore peptide synthetase gene from plant-growth-promoting Pseudomonas putida WCS358.

Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety. Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions. In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene. Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning. This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each. ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans. It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358. PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P. aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P. aeruginosa. It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.

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.

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.

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.

Identification and characterization of a novel nuclear factor of activated T-cells-1 isoform expressed in mouse brain.

The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3'-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context.

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.

Apoptosis induced by crosslinking of CD4 on activated human B cells.

Using immunofluorescence, RT-PCR, and Western blotting, we have demonstrated the ability of human B cells to express CD4. In each of the 10 lymphoblastoid cell lines (LCL) tested there was variable, but definite, proportion of CD4-positive B cells. Expression of CD4 was related to the cell cycle; CD4 was expressed in the G1 phase and continued at later phases of the cell cycle. CD4 was in part internalized and degraded by the LCL B cells. Surface CD4 was associated to lck and its crosslinking resulted in tyrosine phosphorylation. Additional experiments conducted on freshly prepared tonsillar B cells demonstrated that CD4 was expressed by large activated B cells, but not by small resting B cells. However, not all the activated tonsillar B cells had surface CD4 since germinal center cells were CD4-negative. Crosslinking of CD4 on LCL or on tonsillar activated B cells resulted in apoptosis in vitro, a finding that indicates the capacity of CD4 to deliver functional signals to B cells and to play a regulatory function in their physiology. Exposure of CD4 expressing B cells to gp120 under conditions that resulted in CD4 crosslinking also caused apoptosis suggesting some implications for the pathophysiology of AIDS.

Defective recruitment and activation of ZAP-70 in common variable immunodeficiency patients with T cell defects.

We have previously identified a subset of common variable immunodeficiency (CVID) patients with defective T cell function associated with impaired activation of the TCR-dependent tyrosine phosphorylation cascade. Here we have assessed the structural and functional integrity of the principal components involved in coupling the TCR/CD3 complex to intracellular tyrosine kinases in two of these patients. We show that ZAP-70 fails to bind the signaling-competent CD3zeta tyrosine phosphorylation isoform and to become activated following TCR engagement, suggesting that defective recruitment of ZAP-70 might underlie the TCR signaling dysfunction in these patients. Determination of the nucleotide sequences encoding the intracellular domains of the CD3/zeta subunits and ZAP-70 did not reveal any mutation. Furthermore, ZAP-70 from these patients could interact in vitro with recombinant phospho-zeta, ruling out genetic defects at the immunoreceptor tyrosine-based activation motif/SH2 domain interface responsible for ZAP-70 recruitment to the activated TCR. No defect was found in expression, activity or subcellular localization of Lck, which is thought to be primarily responsible for CD3zeta phosphorylation. Hence, while the T cell defect in these CVID patients can be pinpointed to the interaction between ZAP-70 and CD3zeta, the integrity in the components of the signaling machinery involved in this process suggests that additional components might be required for completion of this step.