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Children’s educational products play an important role in enhancing children’s thinking, analytical and judgment abilities, flexibilities and creativity. However, so far there have been no effective assessment criteria for such products. The article proposes an innovative 3-level index system to assess educational products, which comprises 3 first-level indices, 9 second-level indices and some corresponding third-level indices on the basis of pedagogic, psychological and socialogical theories. The system puts forth specific requirements for qualified educational products in terms of educational effects and operating rules, etc., and incorporates the dimension of “age” to make assessment more operable and accurate.

Background Thrombotic thrombocytopenic purpura (TTP) is a life-threatening diagnosis requiring prompt initiation of therapeutic plasma exchange (TPE). Measurement of immature platelet (PLT) fraction (%-IPF) differentiates PLT consumption or destruction from hypoproduction. Study Design and Method Our study evaluated %-IPF changes over the course of TTP treated with TPE and as a measure of treatment efficacy. Eleven idiopathic TTP patients, two human immunodeficiency virus (HIV)-associated TTP patients, and five non-TTP patients with thrombocytopenia were enrolled into our study. All patients were treated with TPE and had ADAMTS13 activity measured. Results All idiopathic TTP patients had a significantly increased %-IPF and decreased absolute immature PLT count (A-IPC) and PLT count at presentation. An A-IPC value of less than 5 × 109/L at presentation has 84.6% sensitivity, 80% specificity, and 91.7% positive predictive value for diagnosing TTP. A concurrent steady decline in %-IPF and increased PLT counts toward normal was observed in TTP patients undergoing TPE. The A-IPC, however, showed an increase and decrease curve that was not seen in the two HIV-associated TTP patients with no response to TPE and the five non-TTP patients. More importantly, reaching an A-IPC ratio of 3 compared to baseline value during TPE can readily differentiate idiopathic TTP from the other two groups and is correlated with good clinical responses to TPE. An abrupt increase of A-IPC during TPE was also noted in a TTP patient who relapsed 3 days before PLT count decrease. A-IPC is positively correlated with ADAMTS13 activity at presentation but negatively correlated with ADAMTS13 activity during recovery. Conclusion A-IPC should be routinely analyzed for diagnosing and monitoring TTP patients.

Genomic analysis has greatly influenced the diagnosis and clinical management of patients affected by diverse forms of hematologic malignancies. Here, we review how genetic alterations define subclasses of patients with acute leukemias, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), non-Hodgkin lymphomas, and classical Hodgkin lymphoma. These include new subtypes of acute myeloid leukemia defined by mutations in RUNX1 or BCR-ABL1 translocations as well as a constellation of somatic structural DNA alterations in acute lymphoblastic leukemia. Among patients with MDS, detection of mutations in SF3B1 define a subgroup of patients with the ring sideroblast form of MDS and a favorable prognosis. For patients with MPNs, detection of the BCR-ABL1 fusion delineates chronic myeloid leukemia from classic BCR-ABL1− MPNs, which are largely defined by mutations in JAK2, CALR, or MPL. In the B-cell lymphomas, detection of characteristic rearrangements involving MYC in Burkitt lymphoma, BCL2 in follicular lymphoma, and MYC/BCL2/BCL6 in high-grade B-cell lymphomas are essential for diagnosis. In T-cell lymphomas, anaplastic large-cell lymphoma is defined by mutually exclusive rearrangements of ALK, DUSP22/IRF4, and TP63. Genetic alterations affecting TP53 and the mutational status of the immunoglobulin heavy-chain variable region are important in clinical management of chronic lymphocytic leukemia. Additionally, detection of BRAFV600E mutations is helpful in the diagnosis of classical hairy cell leukemia and a number of histiocytic neoplasms. Numerous additional examples provided here demonstrate how clinical evaluation of genomic alterations have refined classification of myeloid neoplasms and major forms of lymphomas arising from B, T, or natural killer cells.

TAFRO syndrome is a rare clinicopathologic variant of idiopathic multicentric Castleman disease characterized by Thrombocytopenia, Ascites (anasarca), myeloFibrosis, Renal dysfunction, and Organomegaly. Here, we report a case of TAFRO syndrome in an HIV-negative young Caucasian male who presented with fever, normocytic anemia, thrombocytopenia, and acute renal insufficiency. The serum interleukin-6 (IL-6) level was elevated. Chest and abdominal CT revealed bilateral pleural effusion, ascites, splenomegaly, and multiple mildly enlarged lymph nodes. An excisional biopsy of inguinal lymph node showed a few atrophic follicles and expansion of interfollicular areas by marked vascular proliferation and polytypic plasmacytosis. HHV-8 was negative. Subsequent bone marrow biopsy was normocellular with moderately increased megakaryocytes and occasional megakaryocytic emperipolesis. His signs and symptoms improved after treatment with methylprednisolone and tocilizumab (anti-IL-6 receptor antibody). Our study confirms the distinctive nature of this syndrome, which should allow for better recognition and appropriate therapy. Keywords: Castleman-Kojima disease, TAFRO syndrome, Idiopathic multicentric Castleman disease, IL-6, HHV-8

In order to interpret the physical feature of Bessho form translating-pulsating source Green function, the phase function is extracted from the integral representation and stationary-phase analysis is carried out in this paper. The complex characteristics of the integral variable and segmentation of the integral intervals are discussed in m complex plane. In θ space, the interval [−π/2+φ, −π/2+φ-iɛ] is dominant in the near-field flow, and there is a one-to-one correspondence between the real intervals in m space and the unsteady wave patterns in far field. If 4τ>1 (τ is the Brard number), there are three kinds of propagation wave patterns such as ring-fan wave pattern, fan wave pattern and inner V wave pattern, and if 0

CRABP-II, a retinoic acid binding protein, shuffles retinoic acid from cytoplasm into nucleus and forms a complex with nuclear retinoic acid receptor to facilitate transcriptional activities of retinoic acid. In this study, we studied the expression patterns of CRABP-II in pancreatic ductal adenocarcinoma (PDAC) compared with those in normal pancreas, chronic pancreatitis, and precancerous lesions. We showed no detectable expressions of CRABP-II in normal pancreatic parenchyma, normal ductal epithelium, and chronic pancreatitis. In contrast, the expression of CRABP-II was readily detected in all PDACs including metastatic PDACs. CRABP-II staining was also observed and progressively increased from pancreatic intraepithelial neoplasia 1 to 3. In addition, when fine needle aspiration specimens were evaluated from patients with PDAC, CRABP-II was positive in 55.6% cases if cytology diagnosis was "atypia," and in 87.5% cases, if "malignancy." Our study suggests that CRABP-II is highly and specifically expressed in PDAC and is more commonly expressed in high-grade precursor cancerous lesions than in low-grade lesions. Therefore, overexpression of CRABP-II is a late event of pancreatic carcinogenesis, and it could be used as a diagnostic marker to distinguish PDAC from other benign pancreatic conditions in both resection and cytology specimens.

Clonal heterogeneity and evolution of mantle cell lymphoma (MCL) remain unclear despite the progress in our understanding of its biology. Here, we report a 71-yr-old male patient with an aggressive MCL and depict the clonal evolution from initial diagnosis of typical MCL to relapsed blastoid MCL. During the course of the disease, the patient was diagnosed with classic Hodgkin lymphoma (CHL) and received a CHL therapeutic regimen. Molecular analysis by next-generation sequencing of both MCL and CHL demonstrated clonally related CHL with characteristic immunophenotype and PDL1/2 gains. Moreover, our data illustrate the clonal heterogeneity and acquisition of additional genetic aberrations including a rare fusion of SEC22B-NOTCH2 in the process of clonal evolution. Evidence obtained from our comprehensive immunophenotypic and genetic studies indicates that MCL and CHL can originate from a common precursor by divergent clonal evolution, which may pose a therapeutic challenge.

In this paper some new flocking models are introduced. The contact mechanism between agents is from the famous Cooker-Smile model. In these models, the information exchange in the system is fully connected and the agents can adjust their velocities to form a flock. The important innovation of this paper is the flocking agents will automatic slow their velocities to avoid collision when they detects their relatively velocities so quickly and they are too closed with other agents. Using these models, the information interaction between the agents is completely automated which means they do not need human help to finish the flock. We only need to give the initial velocity for each agents they will complete the flocking and not run into each other. Also, we give the final velocity of the system and from the expression it show this velocity is only depended on their agent's initial velocity.

Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio Conclusion We have established an objective and quantitative MFC method to risk stratify post induction AML patients by risk for relapse, MRD clearance and likelihood of survival. Loss of PDC correlates with residual leukemia, is highly specific for MRD positivity in post-induction patients, and strongly predicts poorer overall survival and higher likelihood of relapse. Loss of PDC also predicts persistent MRD in post-induction MRD-pos. patients despite further therapy, suggesting that MRD-pos. patients with normal PDC may benefit from further therapy prior to transplant, while MRD-pos. patients with loss of PDC may not. Figure 1. Figure 1. Disclosures Goldberg: AROG: Research Funding; Pfizer: Research Funding; Celgene: Consultancy. Geyer:Dava Oncology: Honoraria. Levine:Isoplexis: Equity Ownership; C4 Therapeutics: Equity Ownership; Gilead: Honoraria; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Imago: Equity Ownership; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Epizyme: Patents & Royalties; Janssen: Consultancy, Honoraria. Tallman:BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board.

Introduction Plasmacytoid dendritic cells (PDC) are the major natural type I interferon producing dendritic cells that play critical roles in the immune response. PDC are differentiated from hematopoietic stem cells under the control of multiple transcription factors including GATA-2, IKAROS, IRF8, TCF4, ID2 and RUNX1. PDC can be easily identified by flow cytometry due to high-level expression of CD123, HLA-DR and CD303/BDCA2 in the context of lack of lineage markers. In healthy subjects, PDC are Patients and Methods AML patients were searched from the patient database at the MSKCC between 5/2014 and 12/2017. Flow cytometric data obtained on the bone marrow aspirate at the diagnosis were reviewed and cases with PDC proportion greater than 3% of WBC (defined by CD45 positive cells) were included. All the patients had genomic sequencing studies (a targeted NGS panel comprising 30 genes with mutations enriched in AML) performed at diagnosis. PDC and leukemic blasts were sorted by flow cytometry from a subset of patients (N=6) and mutational profiles were compared after sequencing studies. Histopathologic assessment was also performed. Results 24 patients were identified (20 male and 4 female) with a median age of 69 years old (Interquartile range IQR: 56-76). The prevalence was approximately 5% among total new AML patients diagnosed during the same time period. 21 (88%) patients were secondary AML (18 AML with myelodysplasia-related changes (AML-MRC) and 3 therapy-related AML) and only 3 (12%) were de novo AML. No skin presentation was seen. 17 (71%) patients had cytogenetic abnormalities but only 1 with complex karyotype, 1 del(5p) and 3 del(7q). 14 (58%) had pathogenic RUNX1 aberrations (13 mutations and 1 rearrangement, Figure 1A). Other recurrent mutations included ASXL1 (N=7, 29%), IDH1 (N=4, 17%), SRSF2 (N=4, 17%), DNMT3A (N=3, 13%), TET2 (N=3, 13%), NRAS (N=3, 13%), and TP53 (N=3, 13%). The median of PDC proportion was 8.1% (IQR: 4-12.7%). In contrast to BPDCN, these PDC were negative for CD56, TCL-1 and TdT (Figure 1B). The leukemic blasts from 17 (71%) patients showed cross-lineage antigen expression including CD19 (N=4), CD2 and/or CD7 (N=9), and both CD7/CD19 (N=4). The sorted leukemic blasts and PDC shared identical mutations in 6 examined patients confirming the clonal relationship. Unlike CMML with increased PDC, PDC in AML did not form aggregates, showed leukemic distribution pattern and were also increased in peripheral blood at diagnosis. 17 patients received Daunorubicin based induction therapy and 6 had hypomethylating agents. 13 achieved complete remission (5 MRD negative, 6 MRD positive, 2 unknown MRD status). 11 received allo-HSCT with 5 relapsed post transplant. With a median follow-up of 17 months, 18 patients have died. The median survival was 20 months (Figure 1C). Conclusion AML with plasmacytoid dendritic cell differentiation has unique clinicopathologic features including predominantly secondary AML, enriched for RUNX1 mutations, frequent cross-lineage antigen expression and poor prognosis. The PDC are clonally related to the leukemic blasts. CMML with PDC proliferation, AML with PDC differentiation and BPDCN might represent a spectrum of myeloid stem cell neoplasm with aberrant PDC differentiation. Further studies are needed to investigate the molecular mechanism of particularly the role of RUNX1 in PDC differentiation in AML. Figure 1 Figure 1. Disclosures Goldberg: Celgene: Research Funding; Abbvie: Research Funding; AROG: Research Funding; Pfizer: Research Funding. Tallman:ADC Therapeutics: Research Funding; Orsenix: Other: Advisory board; AbbVie: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; Daiichi-Sankyo: Other: Advisory board; AROG: Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria.

Introduction Secondary acute myeloid leukemia (s-AML) includes AML with myelodysplasia-related changes (AML-MRC) transformed from an antecedent diagnosis of myelodysplastic syndrome (MDS) or from myeloproliferative and myelodysplastic overlap syndrome (MPN/MDS), as well as therapy-related AML (t-AML) arising in patients with prior exposure to leukemogenic therapies. S-AML has a dismal prognosis. Although the genomic profiling of AML has been well studied, few studies have focused on the molecular aberrations of s-AML. A recent study has demonstrated shared genomic aberrations by AML-MRC and t-AML including mutations in TP53, SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2. Another recent study has shown that AML patients with mutations in TP53 and chromatin-splicesosome confer poor prognosis and likely represent s-AML. As such, further characterization of the genomic landscape of s-AML is needed. Recurrent somatic PHF6 mutations are rare in de novo AML (~3%), but the frequency and characteristics of PHF6 mutated s-AML are largely unknown. Patients and Methods AML-MRC and t-AML patients were searched from the patient database at the MSKCC between 1/2014 and 12/2016. All the patients were reviewed and the diagnosis was confirmed. Only patients with genomic sequencing studies (a targeted NGS panel comprising 30 genes with mutations enriched in AML) performed at diagnosis were included. Results 152 patients were identified. There were 7 t-AML patients with favorable cytogenetic abnormalities that likely represent a distinct biological subgroup; therefore these cases were excluded from further analysis. AML-MRC (N=75) and t-AML (N=70) patients had similar clinical characteristics including ages, gender distribution, cytogenetic risk, treatment and survival (median survival: 8 months in t-AML and 10 months in AML-MRC, p=0.4); The mutational profiles of AML-MRC and t-AML cohorts were also comparable with recurrent TP53, PHF6, RAS, and RUNX1 mutations, albeit with different frequencies (Figure 1; Red represents t-AML and blue AML-MRC). Therefore we combined AML-MRC and t-AML cohorts for further analysis. 43 (29.7%) patients had TP53 mutations, 17 (11.7%) patients had PHF6 mutations, and 85 (58.6%) patients had neither mutation (non-TP53/non-PHF6). TP53 and PHF6 mutations were mutually exclusive. TP53 mutations were nearly exclusively associated with adverse cytogenetic abnormalities including alterations involving chromosomes (chr) 5p and/or 7q (41/43, 95.3%) with the majority being complex karyotype (30/43, 70%). By contrast, only 18% PHF6 mutated s-AML patients had chr 5p/7q abnormalities and none of them had complex karyotype. Among the patients with non-TP53/non-PHF6 mutations, 30% had chr 5p/7q abnormalities and 6% had complex karyotype. Although secondary AML has very poor prognosis, PHF6 mutated patients had better overall survival than the other two groups (Log rank test, p=0.0095; median survival: PHF6 vs TP53 vs. non-TP53/non-PHF6: 24 vs. 7 vs. 9 months). Immunophenotypically, the blasts from PHF6 mutated patients showed a high frequency of loss of CD38 (44% vs 17%, p=0.04) and CD33 expression (69% vs 28%, p=0.004) compared to PHF6 unmutated cases, indicative of a less mature, stem-cell derived phenotype Conclusion PHF6 mutations define a unique subset of s-AML that is associated with a more primitive stem/progenitor immunophenotype, absent complex karyotype and relatively better outcomes. PHF6 mutations are mutually exclusive to TP53 mutations. Studies are needed to further define the genomic classification of s-AML patients in an unbiased way and to elucidate the role of PHF6 mutations in s-AML. Figure 1 Figure 1. Disclosures Goldberg: AROG: Research Funding; Abbvie: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Tallman:Orsenix: Other: Advisory board; AbbVie: Research Funding; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Levine:Loxo: Consultancy, Equity Ownership; Gilead: Honoraria; Roche: Consultancy, Research Funding; Epizyme: Patents & Royalties; Imago: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Janssen: Consultancy, Honoraria; Isoplexis: Equity Ownership; Prelude: Research Funding.

In order to retrieve similar trademarks from large-scale trademark databases, combining the characteristics of trademark images, this paper presents a trademark image retrieval method based on regional and border feature fusion. Based on the target image extraction, the proposed approach describes the target region and border features. The region feature description is mainly based on the concept of partition block statistics. The region is divided into equal-area unit using concentric circles, and feature extraction is performed in each small block unit. For the border feature description, this study first detect corners, and then construct a Delaunay graph and extract features by combining the corner detected and the Delaunay triangulation reconstruction. In the search process, the method also incorporates information such as the trademark's color characteristics, trademark classification, and trademark keywords. The present study carried out image retrieval experiment on CE-SHAPE-1 database containing 1400 MPEG-7 core experimental shape, a classification trademark database containing 2000 images, and a national trademark database containing approximately 4.89 million images. The experimental results show that the proposed approach combines the advantages of region and border feature description, and can choose the best among various local optimizations, which makes the retrieval result more effective, more in line with human visual perception, and improves the retrieval accuracy.

Rapid progress has recently been made regarding how phospholipase C (PLC)-β functions downstream of G protein-coupled receptors and how PLC-β functions in the nucleus. PLC-β has also been shown to interplay with tyrosine kinase-based signaling pathways, specifically to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1. In this regard, a new multimolecular signaling platform, named SPS complex, has been identified. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. Furthermore, a growing body of work suggests that PLC-β also participates in the differentiation and activation of immune cells that control both the innate and adaptive immune systems.

This article expands understanding of gender and advancement in academic science by going into a new dimension of inquiry: Focusing on women associate professors in computing, it assesses the relationship between perceived chances for promotion to full professor and indicators of entrepreneurship, as part of key sets of individual and departmental independent variables that are also addressed. Data from a national survey of women in academic computing indicate that time spent in entrepreneurial activity does not predict excellent/good (compared to fair/poor) chances for promotion perceived by these women faculty, nor does the quantity/quality of entrepreneurial activity that they report for their home units. Departmental reward structures reported as favoring entrepreneurial activity negatively predict perceived chances for promotion. Other key individual and departmental characteristics also predict chances for promotion: faculty members’ age, collaboration, family characteristics, departmental climate, and US (compared to Canadian) location. Findings from interviews with a small subset of respondents to the survey illuminate the survey findings on the role of entrepreneurial factors in perceived chances for advancement.

This paper illustrates the significance of the purity of cyclostationary signals and presents a new method for blind extraction of these signals. The advantage of the method can not only extract the cyclostationary signals from low order to high order in turn but also perform algorithm without knowing cyclic frequencies of the extracted signals. The effectiveness of the proposed method is finally demonstrated by computer simulations and experiments.

Src family kinases (SFK) are critical for initiating and regulating the response of mast cells activated by engagement of the high-affinity IgE receptor, FcεRI. Lyn is the predominant SFK in mast cells and has been ascribed both positive and negative roles in regulating mast cell activation. We analyzed the mast cell phenotype of WeeB, a recently described mouse mutant that expresses a Lyn protein with profoundly reduced catalytic activity. Surprisingly, we found that this residual activity is sufficient for wild-type levels of cytokine production and degranulation in bone marrow-derived mast cells after low-intensity stimulation with anti-IgE. High-intensity stimulation of lyn−/− bone marrow-derived mast cells with highly multivalent Ag resulted in enhanced cytokine production as previously reported, and WeeB cells displayed an intermediate phenotype. Under this latter condition, SFK inhibition using PP2 increased cytokine production in wild-type and WeeB but not lyn−/− cells, resulting in substantially higher levels in the PP2-treated WeeB than in lyn−/− cells. Restoration of wild-type and WeeB lyn alleles in lyn−/− cells generated activation phenotypes similar to those in nontransduced wild-type and WeeB cells, respectively, whereas a kinase-dead allele resulted in a phenotype similar to that of empty-vector–transduced cells. These data indicate that inhibition of Lyn and/or SFK activity can result in higher levels of mast cell activation than simple deletion of lyn and that only near-complete inhibition of Lyn can impair its positive regulatory functions. Furthermore, the data suggest that both positive and negative regulatory functions of Lyn are predominantly carried out by its catalytic activity and not an adaptor function.

Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.

Septic transfusion reactions (STRs) resulting from transfusion of bacterially contaminated platelets are a major hazard of platelet transfusion despite recent interventions. Active and passive surveillance for bacterially contaminated platelets was performed over 7 years (2007-2013) by culture of platelet aliquots at time of transfusion and review of reported transfusion reactions. All platelet units had been cultured 24 hours after collection and released as negative. Five sets of STR criteria were evaluated, including recent AABB criteria; sensitivity and specificity of these criteria, as well as detection by active and passive surveillance, were determined. Twenty of 51,440 platelet units transfused (0.004%; 389 per million) were bacterially contaminated by active surveillance and resulted in 5 STRs occurring 9 to 24 hours posttransfusion; none of these STRs had been reported by passive surveillance. STR occurred only in neutropenic patients transfused with high bacterial loads. A total of 284 transfusion reactions (0.55%) were reported by passive surveillance. None of these patients had received contaminated platelets. However, 6 to 93 (2.1%-32.7%) of these 284 reactions met 1 or more STR criteria, and sensitivity of STR criteria varied from 5.1% to 45.5%. These results document the continued occurrence of bacterial contamination of platelets resulting in STR in neutropenic patients, failure of passive surveillance to detect STR, and lack of specificity of STR criteria. These findings highlight the limitations of reported national STR data based on passive surveillance and the need to implement further measures to address this problem such as secondary testing or use of pathogen reduction technologies.

Btk plays crucial roles in the differentiation and activation of B and myeloid cells. Despite drastic reductions of other Ig isotypes, paradoxically high IgE responses have been known inbtkmutant mice. Here we show thatbtk–/–dendritic cells exhibit a more mature phenotype and a strongerin vitroandin vivoT cell-stimulatory ability than wild-type cells. Increased IgE responses were induced by adoptive transfer ofbtk–/–dendritic cells into mice. Consistent with the stronger T cell-stimulatory ability ofbtk–/–dendritic cells,btk–/–mice exhibited enhanced inflammation in Th2-driven asthma and Th1-driven contact sensitivity experiments. These negative regulatory functions of Btk in dendritic cells appear to be mediated mainly through autocrine secretion of IL-10 and subsequent activation of Stat3.

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening diagnosis requiring prompt initiation of therapeutic plasma exchange (TPE). Measurement of immature platelet (PLT) fraction (%-IPF) differentiates PLT consumption or destruction from hypoproduction.Our study evaluated %-IPF changes over the course of TTP treated with TPE and as a measure of treatment efficacy. Eleven idiopathic TTP patients, two human immunodeficiency virus (HIV)-associated TTP patients, and five non-TTP patients with thrombocytopenia were enrolled into our study. All patients were treated with TPE and had ADAMTS13 activity measured.All idiopathic TTP patients had a significantly increased %-IPF and decreased absolute immature PLT count (A-IPC) and PLT count at presentation. An A-IPC value of less than 5 × 10(9) /L at presentation has 84.6% sensitivity, 80% specificity, and 91.7% positive predictive value for diagnosing TTP. A concurrent steady decline in %-IPF and increased PLT counts toward normal was observed in TTP patients undergoing TPE. The A-IPC, however, showed an increase and decrease curve that was not seen in the two HIV-associated TTP patients with no response to TPE and the five non-TTP patients. More importantly, reaching an A-IPC ratio of 3 compared to baseline value during TPE can readily differentiate idiopathic TTP from the other two groups and is correlated with good clinical responses to TPE. An abrupt increase of A-IPC during TPE was also noted in a TTP patient who relapsed 3 days before PLT count decrease. A-IPC is positively correlated with ADAMTS13 activity at presentation but negatively correlated with ADAMTS13 activity during recovery.A-IPC should be routinely analyzed for diagnosing and monitoring TTP patients.

The E2997 Phase III trial included preservation of valuable chronic lymphocytic leukemia (CLL) patient specimens and relevant clinical outcome data. Using a novel high-resolution technology on a flow cytometry platform, we assessed 79 E2997 samples for the expression of 27 analytes that reflected the activity of signaling pathways and apoptosis. We found that the expression levels of ZAP70 segregated the samples into two subpopulations with the distribution showing a peak-trough-peak configuration. Although prior assessment of ZAP70 by standard procedures did not reveal any prognostic information, we found by using the trough in the distribution as a cutpoint that ZAP70 expression levels were significantly correlated with both progression-free survival and overall survival. Additionally, the cells expressing high versus low levels of ZAP70 demonstrated distinct molecular organization as indicated by the other analytes assessed. Our analysis demonstrates the value of ZAP70 expression as a prognostic indicator and suggests that the different clinical results may be due to the distinct molecular biology of the ZAP70-low versus the ZAP70-high CLL samples.

Circulating immature platelet fraction (IPF) reflects real-time thrombopoiesis and correlates with platelet recovery from thrombocytopenic presentations. To understand the dynamics of IPF in platelet transfusions, we quantified the %-IPF in single-donor platelet components (SDP) during prolonged storage. %-IPF significantly increased from baseline by day 5 post-donation. Absolute IPF counts (A-IPC) had similar significant increments. However, gamma-irradiation suppressed the increments of %-IPF and A-IPC by >50%. Ultrastructural analysis of SDP units at day 10 showed well preserved morphology of immature platelets. Our findings suggest that IPF might actively expand ex-vivo and may have a longer shelf life than their mature counterparts. Closer study of IPF may be of critical clinical importance for transfusion practices.

Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation.

Mast cells are major effectors in high-affinity IgE receptor (FcɛRI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-β3 is crucial for FcɛRI-mediated mast cell activation. Plcb3(-/-) mice showed blunted FcɛRI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, FcɛRI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-β3 constitutively interacts with FcɛRI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-β3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, FcɛRI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-β3- and SHP-1-mediated signaling pathway for FcɛRI-mediated cytokine production.

Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)–β3-dependent manner. Thus, PLC-β3–deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)– deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn−/−;PLC-β3−/− mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn−/−;PLC-β3−/− mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr536 and Tyr564 is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr564 by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr536 in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-β3–mediated regulatory mechanism of SHP-1 and Stat5 activities.

Traditionally, it is thought that IgE binding to mast cells via the high-affinity receptor (FcepsilonRI) is simply a passive 'sensitization' step prior to activation by receptor aggregation or cross-linking with multivalent antigen or other cross-linking agents. However, in addition to receptor up-regulation, recent studies have shown that monomeric IgE can induce survival and other activation events including increased histamine content, degranulation, leukotriene release, receptor internalization, adhesion, migration and DNA synthesis. Various IgE molecules exhibit a vast spectrum of heterogeneity: the highly cytokinergic (HC) group of IgEs at an extreme end of the spectrum can induce survival and other activation events very efficiently, whereas poorly cytokinergic (PC) IgEs at the other end can do so less efficiently. All the IgEs tested appear to be capable of inducing receptor aggregation with HC IgEs having a higher capacity to do so than PC IgEs. HC IgEs can promote the production and secretion of various cytokines including the one(s) that can sustain survival in an autocrine and paracrine mechanism. Consistent with receptor aggregation induced by monomeric IgE, other means of receptor aggregation, e.g. IgE+antigen and IgE+anti-IgE, can also induce survival and other events in unique ranges of stimulation intensity.

Antigen-dependent activation of IgE-bound mast cells is critical for immediate hypersensitivity and other allergic disorders. Recent studies have revealed the effects of monomeric IgEs on mast cell survival and activation. Furthermore, IgE molecules exhibit a wide range of heterogeneity in the ability to induce mast cell activation in the absence of antigen. Highly cytokinergic (HC) IgEs can induce a variety of activation events including cell survival, degranulation, cytokine production, and migration, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. Here, we show that culture of bone marrow cells in the presence of monomeric IgEs results in an increased number of mast cells compared with cultures grown without IgE. Furthermore, time in culture required to generate ≥80% pure mast cells is decreased. IgE molecules can directly influence mast cell progenitors to differentiate into mast cells. mRNA expression of several mast cell proteases and mast cell-related transcription factors is higher in mast cells cultured with an HC IgE than those cultured with a PC IgE or without IgE. Expression of early growth response factor-1, a transcription factor that is involved in the production of TNF-α in mast cells, is enhanced in cultures containing high and low concentrations of HC IgE and a high concentration of PC IgE. Consistent with this, expression of TNF-α is higher in mast cells cultured with HC IgE than PC IgE. Therefore, our results suggest that monomeric IgEs, especially HC IgEs, not only promote mast cell development but also modulate the mast cell phenotype.

The proliferation and differentiation of hematopoietic stem cells (HSCs) is finely regulated by extrinsic and intrinsic factors via various signaling pathways. Here we have shown that, similar to mice deficient in the lipid phosphatase SHIP, loss of 2 Src family kinases, Lyn and Hck, profoundly affects HSC differentiation, producing hematopoietic progenitors with increased proliferation, reduced apoptosis, growth factor–independent survival, and skewed differentiation toward M2 macrophages. This phenotype culminates in a Stat5-dependent myeloproliferative disease that is accompanied by M2 macrophage infiltration of the lung. Expression of a membrane-bound form of SHIP in HSCs lacking both Lyn and Hck restored normal hematopoiesis and prevented myeloproliferation. In vitro and in vivo studies suggested the involvement of autocrine and/or paracrine production of IL-3 and GM-CSF in the increased proliferation and myeloid differentiation of HSCs. Thus, this study has defined a myeloproliferative transformation-sensitive signaling pathway, composed of Lyn/Hck, SHIP, autocrine/paracrine cytokines, and Stat5, that regulates HSC differentiation and M2 macrophage programming.

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcepsilonRI). Activated Lyn phosphorylates the FcepsilonRI subunits, beta and gamma, leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcepsilonRI-stimulated hck(-/-) mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck(-/-) cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory events, such as activation of Syk, Btk, JNK, p38, Akt, and NF-kappaB, are substantially reduced in hck(-/-) mast cells. Analysis of lyn(-/-)hck(-/-), lyn(-/-)FcepsilonRIbeta(-/-), and hck(-/-)FcepsilonRIbeta(-/-) cells shows that Hck exerts these functions via both Lyn-dependent and Lyn-independent mechanisms. Thus, this study has revealed a hierarchical regulation among SFK members to fine-tune mast cell activation.

Traditionally, it is thought that IgE binding to mast cells via the high-affinity receptor (FcepsilonRI) is simply a passive 'sensitization' step prior to activation by receptor aggregation or cross-linking with multivalent antigen or other cross-linking agents. However, in addition to receptor up-regulation, recent studies have shown that monomeric IgE can induce survival and other activation events including increased histamine content, degranulation, leukotriene release, receptor internalization, adhesion, migration and DNA synthesis. Various IgE molecules exhibit a vast spectrum of heterogeneity: the highly cytokinergic (HC) group of IgEs at an extreme end of the spectrum can induce survival and other activation events very efficiently, whereas poorly cytokinergic (PC) IgEs at the other end can do so less efficiently. All the IgEs tested appear to be capable of inducing receptor aggregation with HC IgEs having a higher capacity to do so than PC IgEs. HC IgEs can promote the production and secretion of various cytokines including the one(s) that can sustain survival in an autocrine and paracrine mechanism. Consistent with receptor aggregation induced by monomeric IgE, other means of receptor aggregation, e.g. IgE+antigen and IgE+anti-IgE, can also induce survival and other events in unique ranges of stimulation intensity.

Aggregation of the high affinity receptor for IgE (FcepsilonRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcepsilonRI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with FcepsilonRI beta subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcepsilonRI beta. The latter induced much higher levels of FcepsilonRI beta phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with FcepsilonRI beta, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant FcepsilonRI beta showed that FcepsilonRI beta subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcepsilonRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcepsilonRI beta-ITAM.

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcεRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcεRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcεRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcεRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.

Our aim was to compare rectal perception of patients with diarrhea-predominant irritable bowel syndrome (IBS-D), constipation-predominant irritable bowel syndrome (IBS-C), functional constipation (FC), and healthy controls and to evaluate the therapeutic effect of acupoint transcutaneous electric nerve stimulation (TENS). Age- and sex-matched patients (24 IBS-D, 20 IBS-C, and 30 FC) were selected, and 30 volunteers served as healthy controls. Rectal sensory thresholds were evaluated by rectal balloon distension. Short- and long-term acupoint TENS was given respectively. IBS-D patients had significantly lower rectal sensory thresholds of the first sensation of stool, urgency of defecation, and pain than IBS-C or FC patients or healthy controls (P0.05), but there were no differences in rectal sensory thresholds among IBS-C and FC patients and healthy controls. In each group, females had significantly lower rectal sensory thresholds than males (P0.05), but there was no difference between younger (or = 50 years old) and older (50 years old) patients. Short-term acupoint TENS increased rectal sensory thresholds of IBS-D patients. After 2-month acupoint TENS treatment in IBS-D patients, rectal sensory thresholds were significantly increased, stool times and the intensity of abdominal pain were decreased, and psychological scores were relieved to normal. Lowered rectal perception threshold is a hallmark of IBS-D patients. Females have significantly lower rectal sensory thresholds than males. Acupoint TENS is effective to treat IBS-D.

CDX2, a master transcriptional regulator of intestinal cell differentiation and survival, has been used as a marker to indicate colorectal lineage in adenocarcinomas of unknown origin. Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes for adenocarcinomas of unknown origin, but CDX2 expression in pancreatic disease remains unclear. In this study, we systemically and extensively investigated the expression and role of CDX2 in PDAC. We reported that CDX2 expression is weak and heterogeneous is all normal pancreas and chronic pancreatitis. It is largely expressed in epithelial-lining cells of pancreatic ducts including main ducts, inter-lobular ducts, intra-lobular ducts, intercalated ducts and centroacinar cells, but not in acinar cells or islet cells. CDX2 expression is down regulated during the transformation process from PanIN to PDAC. Only one third of PDACs retain some degree of CDX2 expression, and this group of PDACs have reduced median survival time compared to that of CDX2 negative group (308 days vs. 586 days, p = 0.0065). Metastatic PDACs remain similar expression pattern to that of the primary sites. Our study clearly demonstrates CDX2 expression in pancreatic diseases including PDAC, which is practically important when CDX2 is used to establish the primary sites of adenocarcinomas of unknown origin. In addition, our study also provides CDX2 as a prognostic marker for PDAC and implicates an important role of CDX2 in the development of normal pancreas and PDAC.

Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are expressed at high levels in the central nervous system. The physiologic function of these proteins is unknown. Alpha-synuclein has been implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia, as it is highly expressed in the Lewy bodies from both disorders. The expression of α-synuclein in hematopoietic system has been shown in erythroid precursors and megakaryocytes in bone marrow, as well as erythrocytes and platelets in peripheral blood. Moreover, some studies demonstrated the expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression is shown to be higher in PBMCs of individuals with Parkinson's disease compared to healthy controls. In order to study the role of α-synuclein in development of different hematopoietic elements, we compared bone marrow, peripheral blood and lymphoid organs of age and sex-matched α-synuclein knock-out (KO) mice and wild type (WT) animals of the same genetic background (n=10). Flow cytometric analysis of bone marrow elements did not show differences in the percentages and absolute numbers of erythroid, megakryocytic and myeloid lineages (data not shown). However, differential complete blood cell count (CBC) showed statistically significant decrease in red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (Hct) in KO mice compared to WT mice. No difference was noted in other RBC indices (Table 1). However, platelets were smaller in KO mice as measured by the mean platelet volume (MPV). There was no difference in the number of platelets and white blood cell (WBC) counts. There was a significant reduction in the percentage of circulating lymphocytes, and associated increase in the percentage of neutrophils and monocytes in KO mice compared to WT mice, although the difference in the number of lymphocytes did not reach statistical significance (Table 1). Flow cytometric analysis of T lymphocytes in thymus and peripheral lymphoid organs demonstrated marked defect in development of mature T cells. There was a significant increase in the number of double negative thymocytes in KO mice associated with significant decrease in the number of single positive T cells. Furthermore, splenic CD4+ and CD8+ T cells were markedly decreased in KO mice, indicating that α-synuclein is required for T cell development (Table 2). In summary, our findings indicate an absolute requirement for α-synuclein in development of mature T lymphocytes. The underlying mechanism for this function is subject of future studies. Moreover, while α-synuclein-deficiency does not affect the development of myeloid lineage and platelets, lack of this protein is associated with lower number of erythrocytes, suggesting its role in development and/or survival red blood cells. Disclosures: No relevant conflicts of interest to declare.

Introduction Synucleins comprise a family of small proteins that were first identified in normal and neoplastic brain tissues. As a key component of the Lewy body, a-synuclein plays crucial roles in Parkinson disease and other dementias, and mediates neurotransmitter trafficking. The role of a-synuclein in hematopoiesis is largely unknown; however, in the hematopoietic system, a-synuclein is present in megakaryocytes, platelets, erythroid precursors and erythrocytes. In addition, it has also been detected by RT-PCR in monocytes, T-, B-, and NK-cells. Of interest, there is reduced expression of a-synuclein in the megakaryocytes of myeloproliferative neoplasm (MPN), but not normal reactive marrow or myelodysplastic syndrome, suggesting that a-synuclein could play an important role in the pathogenesis of MPN; while there is increased expression in blasts of megakaryoblastic leukemia. In this study, we utilized a-synuclein-/- mice as a model to investigate the role of a-synuclein in hematopoiesis. We identified an unexpected role of a-synuclein in B cell development and maturation. Methods Age- and sex-matched a-synuclein-/- mice and wild type mice (6-week-old; N=10 each group) were purchased from The Jackson Laboratory (Bar Harbor, ME). Bone marrow cells, splenocytes, and lymph nodes were harvested and flow cytometry analysis performed looking at B cell markers. Histological examination of bone marrow, lymph nodes, and spleen were also performed. Results B220lo immature B cells were comparable between WT and KO mice (WT: 2.62±0.30% vs. KO: 3.55±0.67%, Figure1 Aand Table 1); similarly, pre-B cells identified as B220loCD43+ population were comparable between the two groups (Figure 1B). However, when IgD was applied to separate B220hi population into IgD+ and IgD- subsets, circulating B cells (B220hiIgD+ subset), were significantly reduced by 5-fold in KO mice compared to WT (KO: 0.12±0.05% vs. WT: 0.59±0.37%, p=0.02), whereas B220hiIgD- subset representing transitional B cells was similar between WT and KO mice (WT: 1.54±0.22% vs. KO: 2.09±0.70%, Figure1 A and Table 1). Therefore, although early B cell development is not affected, the number of mature B cells in bone marrow is reduced in a-synuclein deficient mice. In spleen, there was a marked reduction in the number of B cells compared to WT: 5.5+1.6% vs. 14.0+1.9%, respectively (Figure 2A and Table 1). The absolute number of B cells was more drastically reduced in KO mice as the total number of splenocytes was only half of that in WT (Figure 2B and data not shown). Histologically, white pulp areas in KO mice were disorganized compared to WT mice (Figure 2B). These results collectively show that the number and distribution of B cells in spleen is regulated by a-synuclein. On the other hand, though the percentage of B cells in lymph nodes was comparable between WT and KO mice, the absolute number of B cells was lower in KO mice and morphologically the lymph nodes from KO mice were smaller than those from WT mice (Table 1, Figure 3A and data not shown). Normal lymph node cortical/ follicular architecture was missing in KO mice compared to WT controls (Figure 3B). The number of follicles in KO mice was 5-fold lower than that WT controls (WT: 5±0.2/HPF vs. KO: 1±0.3/HPF, p=0.001). Conclusion Our data shows that the number and localization of mature B cells in spleen and lymph nodes is in part regulated by a-synuclein. This is the first report to implicate an important role of a-synuclein in B cell development. The mechanism of a-synuclein regulation in B cells is under investigation. Disclosures: No relevant conflicts of interest to declare.

Background ZAP-70 expression has been used as a negative prognostic indicator for patients with chronic lymphocytic leukemia (CLL) (N Engl J Med 2003;348:1764; N Engl J Med 2004;351:893); however, its usefulness as a clinical test has been undermined by technical difficulties relating to the subjective cut-off threshold for positivity. In fact, analyses of samples from CLL patients enrolled in the US Intergroup Phase III trial, E2997, by standard procedures did not result in ZAP-70 (or any other analyte) providing significant prognostic information. We have previously developed a powerful flow cytometric amplification system to assess many important intracytoplasmic molecules including signaling and apoptotic pathway components. We hypothesized that the technology could reveal previously hidden relationships between outcome and molecular expression. Methods We analyzed 79 blood and marrow samples from CLL patients enrolled in the E2997 trial for the expression of ZAP-70 and 28 other analytes. The 28 other analytes included mostly signaling pathway molecules and apoptotic pathway components. Molecular expression levels determined by flow cytometry were analyzed for their relationship to the clinical data. CD19+ B lymphocytes from 42 healthy volunteers were also assessed as a comparison group. Results We found that CLL samples could be clearly stratified by ZAP-70 expression with a distribution having a peak-trough-peak configuration (Figure). In this way, a definitive threshold was obtained by using the trough as the cut-off. Interestingly, ZAP-70 expression in B cells from healthy persons was unimodal and it peaked in the trough of the distribution of the CLL samples (Figure). Using the ZAP-70 expression trough as a cutoff, unlike the E2997 study, we could delineate patient populations with distinct progression-free survival (p < 0.001) and overall survival (p = 0.004). Moreover, by multivariate analysis ZAP-70 expression was found to be an independent variable (p = 0.01) relating to progression-free survival along with 17p deletion and Rai staging. By assessing the expression levels of the other 28 analytes stratified by ZAP-70 expression, we found statistically significant differences in 10 molecules which included phosphoantigens representing activation levels of various signaling pathways and apoptotic pathway constituents. These findings suggest that ZAP-70 stratification defines 2 distinct types of CLL cells. Besides expression, we found highly significant levels of bivariate correlations between expression levels of the molecules examined including over 30 with correlation coefficients of 0.85 or greater. Interestingly, the correlations varied according to ZAP-70 expression. In samples with high levels of ZAP-70 expression, ZAP-70 was correlated with 13 other analytes with r > 0.5 whereas phospho-ZAP-70 was only correlated with 1 other analyte with the 0.5 threshold. Conversely, in samples with low levels of ZAP-70, phospho-ZAP-70 was correlated with 12 other molecules with r > 0.5 whereas ZAP-70 was correlated with no other analytes. An example showing that ZAP-70 was strongly correlated with Mcl-1 in samples with high levels of ZAP-70 but not in samples with low levels is presented below in the figure. The differential intermolecular relationships seen by ZAP-70 stratification support the possibility that ZAP-70 expression defines 2 different types of CLL cells with clinical outcome implications. Conclusions Our study has uncovered highly significant differential expression levels, intermolecular associations, and organization in CLL samples stratified by ZAP-70 expression. Since stratification by ZAP-70 gave significant prognostic information, we propose that our flow cytometric amplification technology permits characterization of the distinct molecular constitution of the 2 types of CLL cells and provides us with a unique opportunity to understand how the composition of leukemic cells translates into clinical outcome. Additionally, these data provide clues for the development of novel therapies. Disclosures: Kaplan: Pathfinder Biotech: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kaye:Pathfinder Biotech: Employment.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Repeated epicutaneous applications of allergen (Dermatophagoides farinae extract) and staphylococcal enterotoxin B induced AD-like skin lesions on mice. We showed that this dermatitis requires mast cells and T cells, but not B cells or eosinophils, for full expression of dermatitis. The clinical severity of dermatitis was correlated with numbers of mast cells. GM-CSF, TSLPR and FcϵRI were required to attain maximal clinical scores. Moreover, high similarity was found in gene expression patterns in allergen-induced lesional skin and human AD skin, supporting the clinical relevance of our mouse model. We have also been studying spontaneously occurring AD-like skin lesions in phospholipase C (PLC)-β3-deficient mice. Mast cells, but not T or B cells, were indispensable for the development of skin lesions. Remarkable abundance of mast cells in the skin of PLC-β3-/- mice was due to the loss of an adaptor function of PLC-β3 to inhibit IL-3-mediated proliferation of mast cells via SHP-1-mediated suppression of Stat5 activity. Consistent with this, increased numbers of mast cells with phosphorylated Stat5 were found in lesional skin of PLC-β3-/- mice. Therefore, this study demonstrates a critical importance of mast cells in AD and suggests that AD development is controlled by elements of the Stat5-activating pathways, as well as other components (PLC-β3 and SHP-1) of the recently discovered multi-molecular complex SPS.

Background and Aim: Serum cancer antigen (CA) 125 elevation has been reported in patients with liver disease, but it is poorly characterized. The present study aimed to evaluate the range of serum and ascitic CA 125 levels in patients with liver cirrhosis and to explore possible factors associated with CA 125 elevation. Methods: A total of 70 patients were studied. Group I consisted of 30 patients with liver cirrhosis with or without ascites. Group II consisted of 30 patients with digestive malignant tumors with or without ascites. Group III consisted of 10 patients with benign ascites. The CA 125 levels were measured in the serum of all patients and also simultaneously in the ascitic fluid of 15 patients. Results: Serum CA125 levels in 80% of (24/30) patients from group I were elevated, particularly in those with ascites, irrespective of the etiology of cirrhosis. Serum CA 125 levels were correlated with Child–Pugh scores (r = 0.38), but not significantly (P = 0.06). All patients from group II with ascites and from group III had elevated serum CA 125 levels, but there was no difference in the serum CA 125 levels between patients with ascites from group I (275 ± 175 U/mL), group II (368 ± 190 U/mL) or group III (396 ± 287 U/mL), nor was there a significant difference in ascitic CA 125 levels (P > 0.05). The levels of serum CA 125 (198 ± 108 U/mL) were lower than, but correlated with that of ascites (460 ± 234 U/mL, r = 0.58, P = 0.026). The elevation of serum CA 125 accompanied by abnormalities of other tumor markers was more common in malignant ascites than in benign ascites (90% compared with 6%, P

Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.

AIM: To summarize the characteristics of patients suffered from primary biliary cirrhosis associated with ulcerative colitis. METHODS: To report a new case and review the literature. RESULTS: There were 18 cases (including our case) of primary biliary cirrhosis complicated with ulcerative colitis reported in the literature. Compared with classical primary biliary cirrhosis, the patients were more often males and younger similar. The bowel lesions were usually mild with proctitis predominated. While ulcerative colitis was diagnosed before primary biliary cirrhosis in 13 cases, the presentation of primary biliary cirrhosis was earlier than that of ulcerative colitis in our new case reported here. The prevalence of primary biliary cirrhosis among patients of ulcerative colitis was almost 30 times higher than in general population. CONCLUSION: Association of primary biliary cirrhosis with ulcerative colitis is rare. It should be considered in the differential diagnosis of hepatobiliary disease in patients with ulcerative colitis, and vice versa.

AIM: To determine the changes of CD8+ T subsets especially CD8+CD28- T regulatory cells in rat model of experimental colitis induced by 2,4-dinitrofluorobenzene (DNFB). METHODS: The rat model of experimental colitis was induced by enema with DNFB. Ten days later, colonic intraepithelial and splenic lymphocytes were isolated from colitis animals (n = 16) and controls (n = 8). The proportion of CD8+ T cells, CD8+CD28+ T cells and CD8+CD28- T regulatory cells were determined by flow cytometry. RESULTS: The model of experimental colitis was successfully established by DNFB that was demonstrated by bloody diarrhea, weight loss and colonic histopathology. The proportion of CD8+ T cells in either splenic or colonic intraepithelial lymphocytes was not significantly different between colitis animals and controls (spleen: 34.6% ± 7.24% vs 33.5% ± 9.41%, colon: 14.0% ± 8.93% vs 18.0% ± 4.06%, P > 0.05). But CD8+CD28- T regulatory cells from colitis animals were significantly more than those from controls (spleen: 11.3% ± 2.26% vs 5.64% ± 1.01%, colon: 6.50% ± 5.37% vs 1.07% ± 0.65%, P < 0.05). In contrast, CD8+CD28+ T cells from colitis animals were less than those from controls (spleen: 23.3% ± 6.14% vs 27.8% ± 9.70%, P = 0.06; colon: 7.52% ± 4.18% vs 16.9% ± 4.07%, P < 0.05). The proportion of CD8+CD28- T regulatory cells in splenic and colon intraepithelial CD8+ T cells from colitis animals was higher than that from controls (spleen: 33.3% ± 5.49% vs 18.4% ± 7.26%, colon: 46.0% ± 14.3% vs 6.10% ± 3.72%, P < 0.005). CONCLUSION: Experimental colitis of rats can be induced by DNFB with simplicity and good reproducibility. The proportion of CD8+CD28- T regulatory cells in rats with experimental colitis is increased, which may be associated with the pathogenesis of colitis.