Mapping infected cell phenotype.

Quantitative modeling of the phenotypic changes in the host cell during the bacterial infection makes it possible to explore an empirical relation between the infection stages and the quantifiable host-cell phenotype. A statistically reliable model of this relation can facilitate therapeutic defense against threats due to natural and genetically engineered bacterium. In the preliminary experiment, we have collected several thousand cell images over a period of 72 h of infection with a 2-h sampling frequency that covers various stages of infection by Francisella tularenesis (Ft). Segmentation of macrophages in images was accomplished using a fully automatic, parallel region growing technique. Over two thousand feature descriptors for the host cell were calculated. Multidimensional scaling, followed by hierarchical clustering, was used to group the cells. Preliminary results show that the host-cell phenotype, as defined by the set of measureable features, groups into different classes that can be mapped to the stages of infection.

The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells.

The serine-threonine kinase Akt/PKB is activated downstream of phosphatidylinositol 3-kinase in response to several growth factor stimuli and has been implicated in the promotion of cell survival. Although both phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) have been implicated in the regulation of Akt activity in vitro, the relative roles of these two phospholipids in vivo are not well understood. Co-ligation of the B cell receptor (BCR) and the inhibitory FcgammaRIIB1 on B cells results in the recruitment of the 5'-inositol phosphatase SHIP to the signaling complex. Since SHIP is known to cleave PIP3 to generate PI 3,4-P2 both in vivo and in vitro, and Akt activity has been reported to be regulated by either PIP3 or PI 3,4-P2, we hypothesized that recruitment of SHIP through FcgammaRIIB1 co-cross-linking to the BCR in B cells might regulate Akt activity. The nature of this regulation, positive or negative, might also reveal the relative contribution of PIP3 and PI 3,4-P2 to Akt activation in vivo. Here we report that Akt is activated by stimulation through the BCR in a phosphatidylinositol 3-kinase-dependent manner and that this activation is inhibited by co-cross-linking of the BCR to FcgammaRIIB1. Using mutants of FcgammaRIIB1 and SHIP-deficient B cells, we demonstrate that inhibition of Akt activity is mediated by the immune cell tyrosine-based inhibitory motif within FcgammaRIIB1 as well as SHIP. The SHIP-dependent inhibition of Akt activation also suggests that PIP3 plays a greater role in Akt activation than PI 3,4-P2 in vivo.

Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP.

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5'-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc.SHIP complex and regulation of SHIP function during T cell receptor signaling.

Differential activation of the tyrosine kinases ZAP-70 and Syk after Fc gamma RI stimulation.

Engagement of the high-affinity IgG Fc receptor (Fc gamma RI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in Fc gamma RI-mediated signaling. Cross-linking of the Fc gamma RI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the Fc epsilon RI gamma subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with Fc epsilon RI gamma after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.

Immunophenotypic differences between putative hematopoietic stem cells and childhood B-cell precursor acute lymphoblastic leukemia cells.

Acute leukemia cells express myeloid, B-lymphoid and T-lymphoid lineage specific antigens. Many acute leukemias express the hematopoietic progenitor cell antigen CD34. Three proposed models of the normal human hematopoietic stem cell include CD34+ Thy-1low Lin-, CD34+ CD38-, and CD34+ HLA-DR-. The patterns of expression of CD34, Thy-1, CD38, HLA-DR, and multiple lineage-specific antigens on 49 consecutive pediatric B-cell precursor acute lymphoblastic leukemia (ALL) cases submitted for immunophenotyping (36 at first diagnosis, 13 at relapse) were analyzed. CD34+ expression was observed in 67% of the cases. CD34+ expression correlated with Thy-1low expression and expression of myeloid antigens (p < 0.001 and < 0.025, respectively). The CD34+ Thy-1low phenotype was observed in 65% of the cases; the CD34+ CD38- or CD34+ HLA-DR- phenotypes were observed in only three cases. Examples of heterogeneous expression of CD34 and Thy-1 were found in six cases, but CD38 expression was always bright and homogeneous in all positive cases. The data from this analysis indicates that the CD34+ CD38- or CD34+ HLA-DR- phenotypes would be more useful than the CD34+ Thy-1low phenotype for distinguishing normal hematopoietic stem cells from leukemic cells in childhood B-cell precursor ALL.