George Shubinsky

PKCθ is required for hemostasis and positive regulation of thrombin-induced platelet aggregation and α-granule secretion

Platelet activation due to vascular injury is essential for hemostatic plug formation, and is mediated by agonists, such as thrombin, which trigger distinct receptor-coupled signaling pathways. Thrombin is a coagulation protease, which activates G protein-coupled protease-activated receptors (PARs) on the surface of platelets. We found that C57BL/6J and BALB/C mice that are deficient in protein kinase C theta (PKCtheta), exhibit an impaired hemostasis, and prolonged bleeding following vascular injury. In addition, murine platelets deficient in PKCtheta displayed an impaired thrombin-induced platelet activation and aggregation response. Lack of PKCtheta also resulted in impaired alpha-granule secretion, as demonstrated by the low surface expression of CD62P, in thrombin-stimulated platelets. Since PAR4 is the only mouse PAR receptor that delivers thrombin-induced activation signals in platelets, our results suggest that PKCtheta is a critical effector molecule in the PAR4-linked signaling pathways and in the regulation of normal hemostasis in mice.

Protein kinase C-theta in platelet activation

Members of the protein kinase C (PKC) family of serine/threonine kinases have been implicated in several physiological processes regulating the activation response of platelets. They are involved in processes leading to granule secretion, integrin activation, platelet aggregation and spreading, and procoagulation. The protein kinase C θ (PKCθ) isoform, which was originally identified in T lymphocytes, is also expressed at relatively high levels in platelets, wherein it is involved in the regulation of hemostasis and thrombosis. Recent studies suggest a role for PKCθ in protease‐activated receptor (PAR)‐, glycoprotein VI (GPVI) receptor‐ and glycoprotein αIIbβ3 integrin receptor‐linked signal transduction pathways. The present review focuses on the latest observations relevant to the role of PKCθ in platelet activation.

A γ/δ T-cell receptor prolymphocytic leukemia and CD4-/CD8- double-negative immunophenotype in a pediatric patient.

T-cell prolymphocytic leukemia is a very rare neoplasm, peaking in the seventh decade. An extensive search failed to find any report of this malignancy in the pediatric population. The malignant cell is morphologically characterized by a high nucleocytopasmic ratio, condensed chromatin, a single nucleolus, and nongranular basophilic cytoplasm. Cells are usually positive for the &agr;/&bgr; and only rarely to the &ggr;/&dgr; T-cell receptors. Most patients follow an aggressive clinical course, only some respond to anti-CD52. We present a 6-year-old boy with T-cell prolymphocytic leukemia. The malignant cells expressed a postthymic immunophenotype (CD4−/CD8−) and positivity for the &ggr;/&dgr; T-cell receptors. The child died after 8 months despite aggressive chemotherapy, anti-CD52, and an allogeneic bone marrow transplant.

Conversion of childhood acute lymphocytic leukemia (L2) with a double t(12;21) to juvenile myelomonocytic leukemia with a novel t(4;11)(p12;q23): a cytogenetic, morphologic, and immunophenotypic study

Here we describe a cytogenetic and flow-cytometric study of a case in which a conversion of childhood acute lymphocytic leukemia (ALL) into juvenile myelomonocytic leukemia (JMML) occurred. A 3-year-old boy diagnosed CALLA+, pre-B-ALL with double t(12;21) (by fluorescence in situ hybridization analysis), was treated as per the BFM protocol. A cytogenetic analysis performed at 17 months into treatment showed no t(12;21) in bone marrow (BM) cells; however, a novel translocation, namely, t(4;11), involving the p12 locus on chromosome 4 and the MLL gene at 11q23 was detected in monocytes. No cytogenetic abnormalities were found either in Epstein-Barr virus-transformed B cells or in phytohemagglutinin-stimulated T-lymphoid cells. Flow-cytometric analysis demonstrated an asynchronous expression of the antigenic determinants in populations of granulocytic and monocytoid cells: 60% of monocytes expressed low levels of CD14, an unusually high level of CD15, and no CD13 or HLA-DR antigens; 74% of myeloid cells expressed no CD13. Our results indicate that the transformation from B-cell ALL to JMML in this case occurred most probably in the granulocyte-erythroid-macrophage-megakaryocyte progenitor cells without involving the lymphoid cell line. To date, the child is 10 months off therapy and asymptomatic, with t(4;11) in only 3% of the cells.

Agranulocytosis at First Presentation of Autoimmune Hepatitis Type-1

Autoimmune hepatitis (AIH) is a progressive, chronic disease of unknown cause with varying presenting symptoms, ranging from no symptoms through nonspecific symptoms to fulminant hepatic failure. Although nonspecific hematologic abnormalities in AIH may occur, a case of agranulocytosis (<100 neutrophils/microL) associated with a flare of AIH and suspected to be of autoimmune origin was recently reported. Increased levels of suppressing cytokines had been previously reported in bone marrow samples of patients with AIH type-1 (AIH-1). These changes could be related to induction of apoptosis or interference with differentiation and proliferation of the myeloid lineage, hence, playing a meaningful role in the pathogenesis of agranulocytosis in patients with AIH-1. Here, we report a patient with agranulocytosis at first presentation of AIH-1. On the basis of the patients diagnostic evaluation, response to administered therapy, and the review of the literature, we suggest several possible mechanisms relating to bone marrow cytokine milieu changes, in addition to autoimmune pathogenesis, that could explain this phenomenon.

Mycophenolate Mofetil Alone and in Combination with Tacrolimus Inhibits the Proliferation of HT-29 Human Colonic Adenocarcinoma Cell Line and Might Interfere with Colonic Tumorigenesis

Background/Aim: Familial adenomatous polyposis (FAP) was found to be completely reversed in a patient treated with mycophenolate mofetil (MMF) and tacrolimus following kidney transplantation. In this preliminary study, we assessed whether MMF and tacrolimus alone or in combination interfere with the cell cycle and proliferation in a human colonic adenocarcinoma cell line and in the colonic polyps of the patient with FAP. Materials and Methods: Human colonic adenocarcinoma HT-29 cells were treated with tacrolimus and MMF alone and in combination at different concentrations. Cell viability and proliferation were assessed using the MTT assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of Ki-67, a marker of mitotic activity, was evaluated in the patients colonic polyps before and under drug treatment. Results: MMF in combination with tacrolimus induced S-phase cell-cycle arrest and markedly inhibited HT-29 cell proliferation. Ki-67 expression in the patients colonic polyps was significantly reduced following combined tacrolimus and MMF treatment. Conclusion: MMF and tacrolimus synergistically inhibited proliferation of a human colonic adenocarcinoma cell line and interfered with the expansion of colonic crypt proliferation in the polyp from a patient with FAP. The results confirm our clinical observation and indicate the possibility of novel approach to therapy of colorectal neoplasia.

Flow cytometry and morphology analysis of bone marrow in a child with brucellosis and hematologic manifestations.

Constitutional symptoms and pancytopenia are occasionally the initial presentation of pediatric brucellosis. Therefore, in endemic areas, in children with pancytopenia, both brucellosis and malignancy should be included in the deferential diagnosis. We report here a child with pancytopenia and hepatosplenomegaly as manifestations of brucellosis in whom bone marrow morphology and flow cytometry data revealed hemophagocytosis, left shift in myeloid cells and activation changes in antigenic properties of T and B lymphocytes and monocytes. The patient had an uneventful and complete recovery after appropriate antibiotic therapy. Our report demonstrates that bone marrow and flow cytometry findings in children with brucellosis may include significant reactive changes in hematopoiesis.