Zeta-chain-associated Protein Kinase 70 Research Articles

LEF1: a highly specific marker for the diagnosis of chronic lymphocytic B cell leukaemia/small lymphocytic B cell lymphoma

AimsChronic lymphocytic B cell leukaemia (CLL)/small lymphocytic B cell lymphoma (SLL) has proven to be not a uniform entity but to consist of various disease subtypes. CLL might also pose...

Discovery of dual ZAP70 and Syk kinases inhibitors by docking into a rare C-helix-out conformation of Syk

The non-receptor tyrosine kinase Syk (spleen tyrosine kinase) is a pharmaceutical relevant target because its over-activation is observed in several autoimmune diseases, allergy, and asthma. Here we report the identification of two novel inhibitors of Syk by high-throughput docking into a rare C-helix-out conformation published recently. Interestingly, both compounds are slightly more active on ZAP70 (Zeta-chain-associated protein kinase 70), which is the kinase closest to Syk in the phylogenetic tree of human kinases. Taken together, the docking pose and experimental results suggest that the higher affinity of the inhibitors for ZAP70 than Syk originates from a more populated C-helix-out conformation in ZAP70. The latter observation is congruent with the 100-fold lower intrinsic activity of ZAP70 than Syk, as the C-helix-out conformation is inactive. The pharmacophore features of DFG-in, C-helix-out compounds are analyzed in relation to DFG-out inhibitors.

Simultaneous presentation of JAK2 V617F mutation-related essential thrombocythemia and B-cell chronic lymphocytic leukemia.

Simultaneous presentation of JAK2 V617F mutation-related essential thrombocythemia and B-cell chronic lymphocytic leukemia.

Retrospective TREC testing of newborns with Severe Combined Immunodeficiency and other primary immunodeficiency diseases

In Manitoba, Canada, the overall incidence of Severe Combined Immunodeficiency (SCID) is three-fold higher than the national average, with SCID overrepresented in two population groups: Mennonites and First Nations of Northern Cree ancestries. T-cell receptor excision circle (TREC) assay is being used increasingly for neonatal screening for SCID in North America. However, the majority of SCID patients in Manitoba are T-cell-positive. Therefore it is likely that the TREC assay will not identify these infants. The goal of this study was to blindly and retrospectively perform TREC analysis in confirmed SCID patients using archived Guthrie cards. Thirteen SCID patients were tested: 5 T-negative SCID (3 with adenosine deaminase deficiency, 1 with CD3δ deficiency, and 1 unclassified) and 8 T-positive SCID (5 with zeta chain-associated protein kinase (ZAP70) deficiency and 3 with inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKKβ) deficiency). As a non-SCID patient group, 5 Primary Immunodeficiency Disease (PID) patients were studied: 1 T-negative PID (cartilage-hair hypoplasia) and 4 T-positive PID (2 common immune deficiency (CID), 1 Wiskott–Aldrich syndrome, and 1 X-linked lymphoproliferative disease). Both patient groups required hematopoietic stem cell transplantation. In addition, randomly-selected de-identified controls (n=982) were tested. Results: all T-negative SCID and PID had zero TRECs. Low-TRECs were identified in 2 ZAP70 siblings, 1 CID patient as well as 5 preterm, 1 twin, and 4 de-identified controls. Conclusions: TREC method will identify T-negative SCID and T-negative PID. To identify other SCID babies, newborn screening in Manitoba must include supplemental targeted screening for ethnic-specific mutations.

A Case-based Review of Chronic Lymphocytic Leukemia

1. Susan J. Leclair, PhD[⇑][1] 1. University of Massachusetts, Dartmouth, MA 1. Address for Correspondence: Susan J. Leclair, PhD, Department of Medical Laboratory Science, University of Massachusetts, 285 Old Westport Road, Dartmouth, MA 02747-2300, sleclair{at}umassd.edu 1. Explain the role of immunochemical testing as a substitute for bone marrow examination in CLL. 2. List the most common immmunophenotype for classic chronic lymphocyte leukemia. 3. Explain the clinical utility of the new prognostic markers for CLL. 4. List the results of the laboratory tests used to routinely evaluate chronic lymphocytic leukemia. INTRODUCTION The diagnosis and treatment of chronic lymphocytic leukemia (CLL) has undergone a profound expansion in scientific and medical understanding in the last decade. Commonly held beliefs of signs and symptoms at time of diagnosis, required laboratory testing, and choices of therapy have all changed. A 34-year old male medical laboratory science (MLS) student tested his own blood as part of an exercise in the use of an automated instrument. His initial CBC is seen in Table 1. Figure 1 shows an oil immersion magnification view of the lymphocytes on his peripheral blood smear. He agreed to see his primary care physician the next day. The repeat CBC was ordered the next day and the results were essentially unchanged. When questioned the physician said that a viral infection could be the cause. The patient later remarked that he had no obvious signs and symptoms although focused questioning brought out a state of fatigue, night sweats, and a mild viral infection that took over 6 weeks to get over. As is typical with many newly diagnosed patients with CLL, he did not have an anemia or thrombocytopenia. Please see Figure 2. Family history includes a father who died from a lymphoproliferative (non-Hodgkin lymphoma) disorder in the 1980s. Some studies have suggested that there may be a familial risk of leukemia in relatives of lymphoproliferative disorder patients.1 At its base definition, CLL is a monoclonal production of greater than 5x109 B lymphocytes/L mature-looking but dysfunctional lymphocytes in the peripheral blood.2 While small… ABBREVIATIONS: CLL – chronic lymphocytic leukemia, IgVH – immunoglobulin heavy chain mutation, ZAP 70 - zeta chain associated protein kinase 70, FISH – fluorescent in situ hybridization, ATM – Ataxia telangiectasia mutated 1. Explain the role of immunochemical testing as a substitute for bone marrow examination in CLL. 2. List the most common immmunophenotype for classic chronic lymphocyte leukemia. 3. Explain the clinical utility of the new prognostic markers for CLL. 4. List the results of the laboratory tests used to routinely evaluate chronic lymphocytic leukemia. [1]: #corresp-1

Discovery of ZAP70 inhibitors by high-throughput docking into a conformation of its kinase domain generated by molecular dynamics

Very few selective inhibitors of the zeta-chain associated protein kinase 70kDa (ZAP70) have been reported despite its importance in autoimmune diseases. Here, to induce a fit of the so-called gatekeeper residue (Met414) and hydrophobic pocket next to it, a potent Janus kinase 2 (JAK2) inhibitor was first docked into the ATP binding site of ZAP70 by structural alignment of the kinase domains. The resulting model of the complex between ZAP70 and the JAK2 inhibitor was then relaxed by an explicit solvent molecular dynamics simulation with restraints on the backbone atoms. High-throughput docking into the induced-fit conformation of ZAP70 generated by molecular dynamics has revealed 10 low-micromolar inhibitors which correspond to six distinct chemotypes. One of these ZAP70 inhibitors has an IC50 of 110nM for JAK2.

ZAP70 expression in regulatory T cells in allergic rhinitis: effect of immunotherapy

Some allergic diseases may be accompanied by inappropriate number or malfunction of regulatory T cells, which seems to be modified by specific immunotherapy (SIT). To assess if immunotherapy affects regulatory T lymphocytes (Tregs) and their expression of zeta chain associated protein kinase (Zap70), which is essential for T cell activation and intracellular signal downstream transduction. A 3-year prospective, placebo-controlled, double-blind trial of grass SIT was conducted. Forty-one patients sensitized to grass pollen with intermittent allergic rhinitis were randomized to receive SIT (n=21) or placebo (n=20) and 15 healthy were included as a control. Concentration of exhaled nitric oxide (ENO), lung function, symptom scores, the subsets of regulatory T cells (CD4(+) CD25hiCD127low) which express ZAP70 and the level of ZAP70 expression in this subset were assessed at baseline and during the treatment period: before the onset, at the height of the pollen season and after the end of the pollen season. The concentration of nitric oxide and the symptom score were significantly higher in allergic rhinitis patients as compared with the control group. Natural allergen stimulation diminished both the numbers of regulatory T cells that express ZAP70 and the expression of Zap70 within these cells. In the second year of treatment, immunotherapy reduced significantly the symptom scores, concentrations of ENO (P<0.01), intensively increased expression of ZAP70 in regulatory T cells (P<0.001) and the percentage of cells that express ZAP70 (P<0.05) at the height of the pollen season. Placebo treatment did not reduce scores, ENO (P>0.05) nor had influence on Zap70 expression (P>0.05). SIT with grass pollen effectively reduces rhinitis severity and affects allergic airway inflammation reflected by reduction of ENO. Beneficial role of immunotherapy may result not only from the induction of Treg numbers but especially from cell activation and restitution of Treg intracellular signal transduction.

Structural and Biophysical Characterization of the Syk Activation Switch

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.

Copper levels in patients with hematological malignancies

BackgroundCopper levels are elevated in cancer patients compared to normal subjects. However, few studies have investigated the relationship between copper and hematological malignancies. Methods84 patients with hematological diseases were studied, along with 50 healthy individuals. Copper was measured by flame atomic absorption spectrometry. The patients were classified to 2 homogeneous groups, acute and chronic hematological neoplasms, respectively. For the patients with acute hematological malignancies, relapse and remission were investigated in relation to serum copper levels. For chronic hematological neoplasms, serum copper was connected either with stable or progressive disease. Zeta-chain-associated protein kinase 70 (ZAP70) and CD38 expression, along with the unmutated VH immunoglobulin genes (IgVH) status were also determined for the 22 chronic lymphocytic leukemia (CLL) patients. Results54 patients with relapse or progressive disease had elevated copper levels (mean value 1.8mg/l), whereas 30 patients either in remission or in stable disease had normal copper levels (mean value 1.01mg/l) (normal range 0.8–1.3mg/l). ConclusionHence, our study indicates that serum elevated copper levels are associated with hematological malignancies either in relapse or in disease progression, whereas normal copper levels are linked with hematological neoplasms in remission or in stable disease. Furthermore, we report for the first time an association between high serum copper levels and several adverse prognostic markers in CLL, such as increased expression of ZAP70 and CD38, along with elevated percentage of unmutated IgVH.

Trisomy 12 CLLs progress through NOTCH1 mutations

Trisomy 12 CLLs progress through NOTCH1 mutations

Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological Synapse Formation

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL.

The prognostic difference of monoallelic versus biallelic deletion of 13q in chronic lymphocytic leukemia.

Fluorescence in situ hybridization can detect genomic abnormalities in up to 80% of cases and provides prognostic information on patients with chronic lymphocytic leukemia (CLL). Although 13q deletion as the sole abnormality has been found to confer a favorable prognosis, there are little data as to whether there is a difference in prognostic value between monoallelic versus biallelic deletion of 13q. The authors reviewed the electronic database for patients with CLL who carried the 13q deletion as the sole abnormality and presented to The University of Texas MD Anderson Cancer Center (MDACC). Untreated patients were separated into 2 groups: those having monoallelic versus those with biallelic deletion of 13q. Using Mann-Whitney, chi-square, and Kaplan-Meier analysis, the baseline quantitative and qualitative variables for each group, along with the time from presentation to MDACC to treatment, were compared. A total of 176 patients were identified; 143 patients had a monoallelic deletion of 13q, whereas 33 patients had a biallelic deletion. The only significantly different values between the groups were albumin (4.5 g/dL vs 4.4 g/dL; P = .01) and zeta-chain-associated protein kinase 70 (ZAP70) expression (1.7% vs 4.8%; P = .010). The median time from fluorescence in situ hybridization analysis to treatment in both the monoallelic and biallelic groups had not been reached (P = not significant). Except for inconsequential differences in albumin and ZAP70 expression, there was no difference in the baseline characteristics between patients with CLL who had monoallelic or biallelic deletion of 13q. In addition, there was no significant difference in endpoints, including time to treatment.

CLLU1 Gene Expression Level As a Prognostic Parameter in Patients with Chronic Lymphocytic Leukemia

Abstract 4603 BackgroundChronic lymphocytic leukemia (CLL) is the most frequent leukemia in the Western world with an annual incidence of 5/100,000. The clinical course of the disease is highly variable; while some CLL patients experience a stable clinical course that will never affect their morbidity or mortality, some of them will eventually progress and require chemotherapy. In addition to the traditional prognostic markers (e.g. Rai and Binet staging systems), more recently, mutational status of the variable regions of the immunoglobulin heavy chains (IgVH), chromosomal aberrations, CD38 expression and Zeta-chain-associated protein kinase 70 (ZAP-70) expression are used to better determine the prognosis in CLL patients. A novel CLL-specific gene, CLL Up-regulated gene 1 (CLLU1) that uniquely overexpressed in CLL patients, was recently demonstrated. It has been shown that CLLU1 mRNA expression levels in CLL patients predict time to initiation of therapy as well as overall survival (OS), and CLLU1 is highly up-regulated in poor-risk groups. The aim of this study is to investigate the relationship between CLLU1 levels and well known prognostic parameters and, to determine the importance of CLLU1 gene on prognosis and clinical course in our CLL patients. Methods116 (46 female, 70 male) CLL patients who consecutively visited our outpatient clinic between May 2009 and March 2010 were enrolled in the study. Median age was 60 years (range, 30–87 years). Blood samples were drawn from the patients for CLLU1 determination, and CLLU1 levels were determined by RT-PCR method. CLLU1 expression level was counted both in CLL patients and healthy B cells as the difference between CLLU1 and abl (taken as an house keeping gene) gene. Then, they are transformed as folds which is the ratio between CLLU1 level in CLL patients and that in healthy B cells. Patients with CLLU1 expression exceeding the CLLU1 expression of normal (CD19+) B-cells were taken as positive. Each patient was followed for at least one year for survival data. For the statistical analysis, student's t-test, Mann-Whitney U test and Pearson correlation were used. p<0.05 was considered as statistical significant. The study was approved by the local research ethics committee, and written informed consent was obtained from the patients. ResultsThere was no relationship between CLLU1 levels and, sex, age, modified RAI and BINET stages, lymphocyte counts and LDH levels at the time of diagnosis. Patients with nodular bone marrow infiltration had lower CLLU1 levels than patients with non-nodular infiltration (57.6 vs 498). Patients with high β2 microglobulin levels had higher CLLU1 levels than the ones with low β2 microglobulin levels (356.7 vs 13.6, p<0.05). ZAP-70 positive patients had higher CLLU1 levels than ZAP-70 negative patients (217.2 vs 10.2, p=0.007) (Figure 1). Among the patients with CD38 levels studied (n=53) CLLU1 levels were higher in patients with CD38 levels above the median value than patients with CD38 levels below the median value (438.4 vs 42.2). CLLU1 levels were higher in cases who needed treatment than cases without treatment. Patients with a shorter time to treatment had higher CLLU1 levels than patients with a longer time to treatment (p=0.028) (Figure 2). [Display omitted] [Display omitted] ConclusionWith a limited number of patients we could demonstrate that CLLU1 levels correlated with β2 microglobulin levels and ZAP-70. Although there were similar findings also with CD38 levels; the association was not statistically significant due to the limited number of cases. Time to treatment was shorter in patients with CLLU1 levels above the median value than patients with CLLU1 levels below the median value. CLLU1 is a promising and specific new prognostic parameter in patients with CLL, and further studies in larger series are needed to define the impact of CLLU1 in the prognosis and clinical course of CLL patients. DisclosureThis study was supported by Istanbul University Research Fund. Disclosures:No relevant conflicts of interest to declare.

Combined normal donor and CLL: Single tube ZAP‐70 analysis

Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index. Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001). The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.

T-cell signalling in antiretroviral-treated, aviraemic HIV-1-positive individuals is present in a raised state of basal activation that contributes to T-cell hyporesponsiveness

Successful antiretroviral therapy (ART) suppresses plasma HIV-1 RNA below detection limits, reducing the chronic insult to the immune systems of infected individuals and supporting a degree of immunological recovery. However, the surface phenotypic profile of T cells in ART-treated patients does not resemble that of healthy, uninfected individuals, but rather shows upregulation of proteins associated with an exhausted immune system. We sought to address whether aviraemic HIV-1 infection, therefore, contributed to long-term alterations in intracellular signalling events within the T cells of infected individuals that contributed to the exhausted phenotype. A flow cytometric approach was employed to assess levels of phosphorylation within T-cell signalling proteins in ART-treated HIV-1-positive patients and HIV-negative individuals. The relative phosphorylation levels of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38, zeta-chain-associated protein kinase 70 (ZAP70), linker of activated T cells, SLP76, nuclear factor kappaB were measured within resting and stimulated CD4(+) and CD8(+) T cells from aviraemic HIV-1-positive and healthy individuals by intracellular staining and flow cytometric analysis. Basal levels of phospho-ZAP70, phospho-ERK and phospho-JNK were two-fold to three-fold higher in HIV-1-positive individuals compared with healthy controls, with phospho-p38 also showing a tendency to increase in HIV-1-positive individuals. Interestingly, in contrast to healthy controls, peripheral blood mononuclear cells from aviraemic, infected individuals were refractory to stimulation with IL-2 and CD3/CD28 showing no enhancement of phosphorylation. CD4(+) and CD8(+) T cells from HIV-1-positive individuals are poorly responsive to direct stimulation through the T-cell receptor due to chronically raised basal activation levels of intracellular signalling molecules.

4.22 Exploring the Clinical Significance of a Positive Direct Antiglobulin Test in CLL: A Community Hospital Experience

Introduction: Autoimmune hemolytic anemia (AIHA) is a common complication in chronic lymphocytic leukemia (CLL), occurring in 10-25% of patients. The direct antiglobulin test (DAT) may be positive at some time during the disease course in up to 35% of cases. There is a controversy regarding the clinical importance of AIHA in CLL. Some studies suggest a negative impact on response rate, progression-free survival (PFS) and overall survival (OS), while others did not confirm these findings. We aim to explore the clinical parameters of a positive DAT test in a cohort of CLL patients diagnosed and treated over a period of 10 years (2001-2011) in a community hospital. Patients-Methods: We studied 152 CLL patients (M/F: 91/61). At diagnosis, median age was 69.4 (34.7-94.6) years, and 65% (99/152), 24% (37/152) and 11% (16/152) of patients were staged as Binet A, B and C respectively. Median follow-up time was 38 (1-138) months. Clinical and biological parameters at diagnosis (full blood count, biochemistry, 2 microglobulin, DAT, zetachain-associated protein kinase 70 [Zap-70] status), treatment and disease outcome were recorded. Results: DAT positivity was detected in 19/152 (12%) patients; 14/19 (74%) patients had this finding at diagnosis while 5/19 patients developed DAT positivity during disease progression. AIHA was diagnosed in 5/152 (3.3%) patients. All 5 patients had positive DAT at diagnosis with 3/5 patients presenting initially with AIHA. AIHIA developed during the course of the disease in 2/5 patients and these episodes were not fludarabine related. DAT positivity at diagnosis was statistically correlated with male gender (p 0.013), higher white blood cell count (p 0.02), higher 2 microglobulin levels (p 0.012), Zap-70 expression (p 0.003) and advanced Binet stage (p 0.002). Patients with positive DAT at diagnosis had a shorter time to CLL treatment, with 77% of them receiving therapy in 1 month postdiagnosis vs 17% of DAT negative patients, p 0.0001). Response rates (complete partial remission) were similar between the two groups. Time to next treatment was not affected by DAT positivity. However, patients with DAT positivity had a lower probability of 5-year survival compared with DAT negative patients (35.6% vs. 81.5%, p 0.048). In multivariate analysis, advanced Binet stage, Zap-70 expression and high 2 microglobulin levels were independent predictors of shorter OS. Positive DAT at diagnosis was statistically important for shorter OS in univariate analysis only (p 0.048). Conclusion: Our findings confirm that DAT positivity at diagnosis of CLL is correlated with negative prognostic factors such as advanced Binet stage, Zap-70 expression and high 2 microglobulin levels. In this study, DAT-positive patients had a lower probability of survival compared with DAT-negative patients. However, DAT status was not proven to be an independent predictor for shorter survival.

Torque Teno Virus 10 Isolated by Genome Amplification Techniques from a Patient with Concomitant Chronic Lymphocytic Leukemia and Polycythemia Vera

An infectious etiology has been proposed for many human cancers, but rarely have specific agents been identified. One difficulty has been the need to propagate cancer cells in vitro to produce the infectious agent in detectable quantity. We hypothesized that genome amplification from small numbers of cells could be adapted to circumvent this difficulty. A patient with concomitant chronic lymphocytic leukemia (CLL) and polycythemia vera (PV) requiring therapeutic phlebotomy donated a large amount of phlebotomized blood to test this possibility. Using genome amplification methods, we identified a new isolate (BIS8-17) of torque teno virus (TTV) 10. The presence of blood isolate sequence 8-17 (BIS8-17) in the original plasma was confirmed by polymerase chain reaction (PCR), validating the approach, since TTV is a known plasma virus. Subsequent PCR testing of plasmas from additional patients showed that BIS8-17 had a similar incidence (~20%) in CLL (n = 48) or PV (n = 10) compared with healthy controls (n = 52). CLL cells do not harbor BIS8-17; PCR did not detect it in CLL peripheral blood genomic deoxyribonucleic acid (DNA) (n = 20). CLL patient clinical outcome or prognostic markers (immunoglobulin heavy chain variable region [IGHV ] mutation, CD38 or zeta-chain associated protein kinase 70 kDa [ZAP-70]) did not correlate with BIS8-17 infection. Although not causative to our knowledge, this is the first reported isolation and detection of TTV in either CLL or PV. TTV could serve as a covirus with another infectious agent or TTV variant with rearranged genetic components that contribute to disease pathogenesis. These results prove that this method identifies infectious agents and provides an experimental methodology to test correlation with disease.

Cyclic nucleotide phosphodiesterase 7B mRNA: An unfavorable characteristic in chronic lymphocytic leukemia

A cost- and time-efficient means to define the prognosis of patients with chronic lymphocytic leukemia (CLL) is desirable but does not yet exist. On the basis of the evidence that CLL cells have enhanced expression of the cyclic nucleotide phosphodiesterase isoform 7B (PDE7B), we hypothesized that PDE7B expression might provide such information. We assessed PDE7B mRNA expression using quantitative real-time PCR in peripheral blood mononuclear cells isolated from 85 patients and 30 normal subjects. We compared PDE7B mRNA expression with that of other disease features to determine if its expression correlates with the prognosis of patients with CLL. We found that CLL patients with PDE7B mRNA levels in the top quartile (greater than ninefold elevation relative to normal controls) have a several-year shorter median time-to-treatment (TTT, 36 months) compared to that of patients whose CLL cells express lower levels of PDE7B mRNA (TTT, 77 months, p=0.001). High PDE7B mRNA expression correlates with expression of zeta-chain-associated protein kinase 70 (ZAP-70), unmutated immunoglobulin heavy chain variable (IGHV) region genes and β2 microglobulin (β2M), but use of a multivariate Cox model revealed that high PDE7B mRNA expression independently predicts a short TTT, even after adjusting for several other disease characteristics (ZAP-70 or CD38 expression, IGHV mutation status and Rai status). High expression of PDE7B is an unfavorable characteristic in CLL. Assessment of PDE7B mRNA expression thus appears to be a clinically useful biomarker to define the prognosis of patients with CLL.

T-Cell Signaling Microcluster Transport Toward the Synaptic Center is Influenced by Membrane Ligand Mobility

The engagement of the T cell antigen receptor (TCR) by antigens induces dynamic molecular reorganization and leads to the recruitment to the plasma membrane of many critical effectors, including zeta-chain associated protein kinase of 70kDa (ZAP70) and adaptor SH2 domain containing leukocyte phosphoprotein of 76kDa (SLP76). Live-cell tracking experiments have revealed the dynamic segregation of these signaling molecules and the formation of immunological synaptic (IS) at the cell-cell interface. However, the correlation between microcluster movement and membrane ligand mobility has remained unclear. Studies so far have focused on either extremely mobile ligands on single-component bilayer surfaces or completely immobile stimuli on glass coverslips. We prepared a supported lipid bilayer system composed of a binary mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) that allows us to vary ligand mobility by adjusting the mixing ratio of phospholipids. Biotinylated anti-human CD3 antibodies are adsorbed via neutravidin to supported lipid bilayers containing polyethylene glycol biotinylated lipids. Human Jurkat T cells are thus activated by our stimulatory bilayer and we track microclusters via fluorescence imaging. We found that ZAP70 microclusters exhibit faster tracking velocity and longer trajectory length in response to increasingly mobile ligands. In contrast, SLP76 microclusters are less responsive to varied ligand mobility. Our measurements of the radial microcluster velocity suggests both ZAP70 and SLP76 microclusters exhibit acceleration toward the center of the cell. Our measurements may help understand the physiological relevance of T-cell synapse dynamics. Correction et al.Biophysical JournalMay 04, 2011In Brief3239-Pos. Full-Text PDF Open Archive

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

<i>NOTCH1</i> PEST domain mutation is an adverse prognostic factor in B‐CLL