Neutrophil Platelet Satellitism Revisited: Sidedness and Domain of Neutrophil Associated Platelet Aggregates.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4469-4469
Author(s):  
Cannon Milani ◽  
Fred J. Schiffman ◽  
Peter J. Quesenberry
Keyword(s):  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Incidental Finding ◽  
Natural Antibodies ◽  
Polymorphonuclear Neutrophils ◽  
Platelet Glycoprotein ◽  
Clear Correlation ◽  
Platelet Aggregates ◽  
Platelet Satellitism

Abstract Abstract 4469 Platelet Satellitism surrounding polymorphonuclear neutrophils has been observed almost exclusively in EDTA-treated blood at room temperature. The mechanism underlying this phenomenon is not fully understood. In a PubMed search of the English language medical literature, there are only 44 reported cases involving the phenomenon of Platelet Satellitism. We report a case of platelet rosetting around neutrophils in a 78-year old woman with incidental thrombocytopenia. Her isolated thrombocytopenia was not mediated by any form of immunosuppression, medications, hypersplenism, intravascular consumption, or diminished platelet production. A diagnosis of Pseudothrombocytopenia was made based upon her peripheral blood smear revealing platelet aggregates displaying sidedness in relation to her neutrophils. Platelet Satellitism is postulated to represent an immunologic phenomenon caused by the presence of natural antibodies in which the platelets aggregate around polymorphonuclear neutrophils. The reasons behind why certain individuals possess agglutinating antibodies that lead to platelet clumping, and others have antibodies that cause platelet satellitism is unknown. A proposed mechanism is natural antibodies directed against different epitopes on the platelet GPIIb-IIIa complex. The reported frequency of platelet Satellitism is much lower than that of EDTA platelet clumping (approximately 1:30,000 blood counts) according to the reviewed literature. Platelet Satellitism to polymorphonuclear neutrophils was initially documented by Field and MacLeod in 1963, and has since been reported as an incidental finding in peripheral blood smears when EDTA was used as an anticoagulant. The process by which platelets bind and form rosettes around polymorphonuclear leukocytes is due to activation of EDTA-dependent antiplatelet and antineutrophil IgG autoantibodies directed against the platelet glycoprotein IIb/IIIa complex and Fc receptors of neutrophils. Further, it is theorized that a non-immunologic cause may play in role in which adherence is induced by thrombospondin or the alpha-granule protein of other platelets. In rare instances, platelets may aggregate around monocytes or basophils. Our retrospective review underscores the importance of recognizing the principle of Pseudothrombocytopenia due to EDTA-induced Platelet Satellitism. This entity is in vitro phenomena which has no clinical bearing in terms of a predisposition to increased mucous membrane bleeding. As in other literature cases, a clear correlation between the presence of IgG antibodies and a specific clinical situation, disease, or use of drugs could not be demonstrated. Therefore, these antibodies, which are present in some normal individuals, might occur naturally. Due to the exposure of certain antigenic structures present on EDTA-modified platelets and neutrophils, they may manifest themselves by triggering the Platelet Satellitism phenomenon. Disclosures: No relevant conflicts of interest to declare.

The Selective Polymorphonuclear Neutrophils (PMN) Degranulation as the Probable Additional Mechanism of the Hemodialysis (HD)-induced Complement Activation

1981 ◽  
Vol 4 (4) ◽  
pp. 174-177 ◽  
Author(s):  
H. Wysocki ◽  
R. Czarnecki ◽  
B. Wierusz-Wysocka ◽  
A. Wykrȩtowicz ◽  
K. Wysocki ◽  
...  
Keyword(s):  
Complement Activation ◽  
Peripheral Blood ◽  
Complement Component ◽  
Polymorphonuclear Neutrophils ◽  
Endothelial Surface ◽  
Artificial Surface ◽  
A Cell ◽  
By Products ◽  
Nylon Wool

The intracellular lysozyme and beta-glucuronidase contents were estimated in PMN isolated from peripheral blood before HD and those from the first portion of full blood leaving the dialyser. The lysozyme estimations were done by the use of the turbidimetric method and beta-glucouronidase was assayed by measuring the release of phenolphtalein from its beta-glucuronate. The cells leaving the cellophane dialyser showed the significantly decreased lysozyme contents while the estimated intracellular activity of beta-glucuronidase was practically equal in both evaluated samples. The results seem to indicate that the HD-associated complement activation may result not only from the simple plasma-cellophane ineteraction. The direct cleavage of the inactive C5 complement component by products released from the PMN specific granules seems to play an important role too. For several years it has been known that the hemodialysis (HD)-induced granulopenia is the result of the pulmonary polymorphonuclear neutrophils (PMN) sequestration (Toren et al., 1970). Recent studies have established that this phenomenon is the consequence of complement activation leading to PMN aggregation with subsequent pulmonary vascular embolization and/or PMN adherence to endothelial surface (Craddock et al. 1977a, Craddock et al., 1979). It was also observed that the contact of patients plasma or plasma obtained from experimental animals with dialyser cellophane results, both in vitro and during HD, in the appearance of the activities: inducing the PMN aggregation, chemotactic and augmenting the PMN adherence (Craddock et al. 1977b, Wysocki et al. 1980). At least the two first are identified as related to the presence of the C5a complement component (Craddock et al., 1977b, O'Flaherty et al., 1979). Similarly, the disseminated complement activation appears to be the main reason of granulopenia accompanying the filtration leukapheresis (FL), the procedure suitable for PMN collection from donors peripheral blood (Hammerschmidt et al., 1978). The PMN are there sequestered in nylon wool layer and subsequently eluted for transfusion purposes. The exposition of blood to artificial surface leads there, like during HD, to the complement (C5) activation. Although, as reported by Wright et al.) 1978b), when plasma and leukocyte enriched blood were diverted by a cell separator before passage through nylon wool filters during FL, complement activation was evident only in plasma from blood containing leukocytes, indicating that extracorporeal complement activation during FL is dependent in large part upon the interaction of leukocytes with nylon wool. The more precise studies of these authors (Wright et al., 1978a) may imply, that the PMN granule constituents released after the previous adherence of cells to nylon fibers are responsible for the FL-associated complement activation. The following studies were done in order to evaluate if the PMN degranulation may take part also in complement activation during hemodialysis carried out with the use of cellophane equipment.

Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3895-3895
Author(s):  
Yair Herishanu ◽  
Inbal Hazan-Hallevi ◽  
Sigi Kay ◽  
Varda Deutsch ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1169-1169
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Yorky Tsin Sik Wong ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

Downregulation of SATB1 Increase the Invasiveness of Jurkat Cell Via Activation of the WNT/Beta-Catenin Signaling Pathway in Vitro

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4825-4825
Author(s):  
Xiaodan Luo ◽  
Lihua Xu ◽  
Dan Liu ◽  
Yaya Wang ◽  
Xiaohong Wu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Peripheral Blood ◽  
Jurkat Cell ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Control Group ◽  
Beta Catenin ◽  
Normal Controls

Abstract Backgroud: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types. Many studies suggest that SATB1 overexpression is an indicator of poor prognosis in various cancers, such as breast cancer, malignant cutaneous melanoma, liver cancer, etc. However, their expression patterns and function values for adult T-cell leukemia (ATL) are still largely unknown. Objective: The aim of this study is to examine the levels of SATB1 in ATL and to explore its function and mechanisms in ATL. METHODS: 20 ATL peripheral blood samples and 20 normal controls were collected. Expressions of SATB1 in both groups were evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cellular proliferation and invasion of SATB1-knockdown Jurkat cells and cells in control group were evaluated by manually count and transwell matrigel invasion assay, respectively. RESULTS: SATB1 expressions were decreased in ATL peripheral blood mononuclear cells (p<0.001) compared with normal controls. Knockdown of SATB1 gene might increase Jurkat cell invasiveness through the activation of Wnt/β-Catenin signaling pathway. CONCLUSIONS: SATB1 expression is down-regulated in ATL and decreased expression of SATB1 increase the invasiveness of Jurkat cell through the activation of Wnt/β-Catenin signaling pathway in vitro. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (81200399) and Key Clinical Disciplines of Guangdong Province (20111219) Disclosures No relevant conflicts of interest to declare.

Androgens MAY BOOST Hematologic Response to ANTI-Thymocyte Globulin in Acquired Aplastic Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3900-3900 ◽  
Author(s):  
Sabrina Giammarco ◽  
Maria Teresa van Lint ◽  
Teresa Lamparelli ◽  
Francesca Gualandi ◽  
Carmen Di Grazia ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Liver Function Tests ◽  
High Dose ◽  
Unrelated Donor ◽  
Gradual Improvement ◽  
Androgen Treatment

Abstract Background: Androgens have shown encouraging results in the treatment of marrow failure, both in the animal model (Nouv Rev Fr Hematol. 1984, 26;391) and in patients with aplastic anemia (Blood. 1990;76: 2222). A first trial comparing ATG with or without androgens , failed to show improved response and survival in the androgens group (Blood. 1985; 66:184). However, in a second randomized trial , females receiving ATG+androgens had significantly superior response rates (BJH 1993,83:145). High dose ATG (twice the conventional dose) and androgens, was given to 87 patients, with 77% response rate and 78% survival at 5 years (Ann Hematol 2006;85:711). More recently sex hormones have been shown to increase telomerase activity, resulting in elongated telomeres in vitro [ Blood. 2009 114:2236), and in vivo [ N Engl J Med. 2016 19;374]. Aim of the study: The aim of this study was to evaluate the outcome of androgen treatment in patients with Aplastic Anemia (AA) who had received a previous course of ATG and were persistently cytopenic. Patients and methods: 78 AA patients were selected as having received oral testosterone 40 mg/day 5-7 days/week , because of persistent cytopenia, in 1-3 cell lines. Typically testosterone was added to the ongoing treatment with cyclosporine. Median age was 37 years (9-72) ; there were 28 males and 50 females.. The median Hb level at start of testosterone, was 8.8 gr/dl( 3-14), the median neutrophil counts 1.7 x10^9/L (0.0-8) and the median platelet count 27x10^9/L (1-128). The median interval between ATG treatment and testosterone was 5 months (0-148); the median duration of androgen treatment was 517 days (range 122- 8984) . Patients were classified as non responders ( no hematologic improvement and/or transfusion dependent), partial responders (transfusion independent and improved peripheral blood counts), or complete responders (normalized peripheral blood counts). Result: Androgen treatment , at this relatively low dose, was well tolerated, with no abnormalities in liver function tests, and no apparent virilization. One patient discontinued testosterone because of intolerance (1,28%). Of the 78 patients treated, 33% (n=23) were classified as non responders, 29% (n=20) as partial responders and 38% (n=26) as completer responders. The median hemoglobin level at 90, 180 and 365 days after androgen therapy were respectively: 9.1; 10.6 and 11.4 g/dl; the median neutrophil counts were respectively 2.0; 2.0 and 3.0 x10^9/L; the median platelets were respectively 38, 54, 75 x10^9/L. There was therefore a gradual improvement of peripheral blood counts with time.With a median follow up of 871 days (range 27-8392), the actuarial 5 year survival, free from transplantation or a second course of immunosuppressive therapy, is 85% (Fig.1); it is 96% for responders and 45% for non responders (Fig2); fifteen out 23 non responders were allografted. Conclusion: The addition of androgen to cyclosporine , improved peripheral blood counts in 67% of our patients, who had failed, or partially failed first line therapy with ATG, with low or absent detectable toxicity. Our results confirm a role for androgens in patients with marrow failure, and this may be relevant for patients eligible for an unrelated transplant, or for older patients discussing an HLA identical sibling transplant. A 3 months course of androgens is still less hazardous as compared to an unrelated donor graft, and may offer the patient an opportunity to achieve transfusion independence without a graft. Disclosures No relevant conflicts of interest to declare.

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

2005 ◽  
Vol 98 (4) ◽  
pp. 1414-1419 ◽  
Author(s):  
Antonino Coppola ◽  
Ludovico Coppola ◽  
Liliana dalla Mora ◽  
Francesco M. Limongelli ◽  
Antonio Grassia ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
B Lymphocytes ◽  
Platelet Activation ◽  
Critical Role ◽  
Strenuous Exercise ◽  
Platelet Glycoprotein ◽  
Vigorous Exercise ◽  
Platelet Aggregates

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

scholarly journals The Flavonoid Wogonin Reduces CLL Cell Survival in Vitro and Leukemia Development in Eµ-TCL1 Mice By Targeting Aberrant TNF Receptor Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1966-1966
Author(s):  
Claudia Dürr ◽  
Bola Hanna ◽  
Fabienne McClanahan ◽  
Andrew James Clear ◽  
Thorsten Zenz ◽  
...  
Keyword(s):  
B Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
In Vivo Studies ◽  
Disease Stage ◽  
Spleen Weight ◽  
Leukemia Development ◽  
Tnfα Receptors

Abstract The importance of the tumor microenvironment in the development of B-cell chronic lymphocytic lekuemia (CLL) is now widely accepted. Previous studies within our and other groups revealed the establishment of an inflammatory milieu in CLL characterized by enhanced expression and secretion of several cytokines and their receptors. Using 250 CLL serum samples and 50 age-matched controls, we found significantly increased levels of the TNFα receptors TNFR1 and TNFR2 in the patients that positively correlated with an adverse clinical outcome. Based on these findings we aimed to investigate the functional role of TNFR signaling in CLL development. In contrast to healthy B cells that do not express TNFα receptors, we detected TNFR1 expression in CLL cells upon survival-inducing culture conditions and in lymph node sections of 80 CLL patients, where TNFR1 signals co-localized to the B cell marker CD20 and were mainly present within proliferation centers. These findings were confirmed in Eµ-TCL1 mice, a well-established mouse model for CLL. Here, CLL cells in the peripheral blood were negative for TNFR1. However, the cells upregulated the receptor upon entering the spleen where they get into contact with accessory cells, receive survival stimuli and undergo proliferation. In addition, increased levels of soluble TNFR1 in the serum were confirmed in the mice. After ligand binding, TNFR1 can activate two different signaling pathways: (1) the extrinsic apoptosis cascade through its death receptor domain, or (2) survival-inducing NFκB signaling. The latter pathway can be blocked by wogonin, a naturally occurring monoflavonoid. In a multitude of in vitro and in vivo studies, wogonin has been shown to exert anti-oxidant, anti-inflammatory and anti-tumor activities. By ex vivo treatment of CLL cells with TNFα we observed NFκB activation which was reversed by TNFα-blocking antibody, suggesting TNFR1-mediated survival signaling in CLL. Therefore, we aimed to test whether wogonin can prevent this signaling in CLL cells and might therefore represent a novel potential drug for CLL. In CLL cocultures, wogonin treatment resulted in a concentration-dependent apoptosis induction, which was significantly increased by the addition of TNFα. To test the effects of wogonin in vivo, we transplanted isogenic, immunocompetent wild-type mice with CLL cells from leukemic TCL1 animals and treated them daily with 40 mg/kg wogonin. When treatment was started 2 days after transplantation, wogonin significantly reduced spleen weights and lead to a reduced CLL content in the spleen, the bone marrow and the peritoneal cavity. If treatment was started in advanced disease stage, wogonin slightly lowered spleen weight and the CLL content in the spleen, whereas the percentage of CLL cells in the peripheral blood was increased. Interestingly, here wogonin treatment resulted in a loss of cell surface TNFR1 expression in splenic CLL cells and increased TNFR1 levels in the serum. These data suggest that wogonin induces a redistribution of CLL cells in vivo, preventing their homing to lymphoid organs and that loss of TNFR1 expression might be involved in this process. In summary, our results show that TNFR1 signaling is involved in CLL cell activation and survival. Targeting this pathway with wogonin reduces CLL cell viability in vitro and leukemia development in TCL1 mice. Disclosures No relevant conflicts of interest to declare.

Leukocyte Integrin αXβ2 Is the Principal Receptor for Candida Albicans on Macrophages and Is Essential for Antifungal Host Defense.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1493-1493
Author(s):  
Samir Jawhara ◽  
Dmitry Soloviev
Keyword(s):  
Candida Albicans ◽  
Conflicts Of Interest ◽  
Fungal Infections ◽  
Critical Role ◽  
Polymorphonuclear Neutrophils ◽  
Cell Mediated Immunity ◽  
Cellular Activation ◽  
Opportunistic Fungal Pathogen ◽  
Antigen Presenting

Abstract Abstract 1493 The induction of cell-mediated immunity to pathogens is the prime task of cells of the innate immune system. Integrin αMβ2 (Mac-1, CR3, CD11b/CD18) is expressed on polymorphonuclear neutrophils (PMNs), monocytes, macrophages (MFC) and NK cells and has been implicated in diverse responses of these cells to infection, including phagocytosis, cell-mediated killing, chemotaxis and cellular activation. In PMNs integrin αMβ2 serves as the principal receptor involved in recognition of the common opportunistic fungal pathogen Candida albicans, and this integrin recognizes the fungal mannoprotein pH-regulated Antigen 1 (Pra1). Professional antigen-presenting cells of myeloid lineage MFC and dendritic cells express integrin αXβ2 (p150,95, CD11c/CD18) which is highly homologous to Integrin αMβ2, but the role of αXβ2 in the immune response to bacterial and fungal infections remains unclear. Employing αM - and αX-KO mice and mutated strains of C. albicans in a model of murine systemic candidiasis, we first demonstrate that mice deficient in αXβ2 exhibit significantly increased susceptibility to the invasive fungal infection. C. albicans induced lethality in αXβ2-KO mice more rapidly (2- to 3-fold faster) than the same inoculum in wild-type mice. MFC derived from mice deficient in α×β2 have a reduced ability to kill or phagocyte C. albicans. When the fungus was injected into mice, 6 hrs after peritoneal of thioglycollate, neutrophil recruitment to C. albicans showed a 20%-30% clearance of the fungus in the αMβ2-KO murine intraperitoneal lavages and a 50–60% rise in the αXβ2-KO lavages, which is consistent with our prior data showing αMβ2-dependency for PMNs-mediated antifungal defense activity. In contrast, when the fungus was injected into the peritoneal cavity 16 hrs after thioglycollate, when most recruited leukocytes are MFC, the C. albicans injected into αXβ2-KO mice showed a 23%-25% clearance of the fungus from the lavages while fungal clearance in αMβ2-KO lavages increased by only 30–40%. Thus, MFC antifungal activity is αXβ2-dependent. These results were further confirmed by in vitro fungicide assays employing isolated murine peritoneal PMNs and MFC. Fungal survival was greatest with PMN derived from αMβ2-KO mice and MFC derived from the αXβ2-KO mice-derived MFC. Disruption of the PRA1 gene of C. albicans, which is the primary ligand for αMβ2, had no effect on MFC fungicide activity, which suggests that αXβ2 recognizes a C. albicans ligand different from Pra1. Taken together, these results suggest that αXβ2 is a principal receptor for C. albicans on macrophages and plays critical role in host antifungal defense in a Pra1-independent mechanism. Disclosures: No relevant conflicts of interest to declare.

Cytogenetics and spectral Karyotyping analysis in CLL Patients: Correlation with FISH and ZAP70 Expression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4844-4844
Author(s):  
Fabio Morato Oliveira ◽  
Daniel Mazza Matos ◽  
Lorena Lobo Figueiredo Pontes ◽  
Belinda Pinto Simoes ◽  
Eduardo M. Rego ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Cytogenetic Analysis ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Karyotype Analysis ◽  
Calf Serum ◽  
Normal Karyotype ◽  
Fish Analysis ◽  
Interphase Cell

Abstract Abstract 4844 Cytogenetic abnormalities play an important role as prognostic factors in CLL. However, due the low mitotic index of CLL B cells in vitro, analysis of a set of subjects for the most commonly known aberrations is usually done by FISH on interphase cell. The objectives of this investigation were the use of the oligonucleotide DSP30 in combination with IL-2, as a B-cell mitogen for cytogenetic investigation in CLL and correlation among the karyotype analysis obtained (G-banding + SKY), FISH profile from unstimulated cells, ZAP70 expression and stratification status for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. Additionally, one set of cell culture was performed for each patient without any stimulant agent, for FISH analysis. The FISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>2.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction. The ZAP70 profile was obtained by flow cytometry analysis. In concordance with literature, the cut off value adopted for ZAP70 was 20%. In a group of 64 subjects studied, the cytogenetic analysis showed chromosomal aberrations in 52 patients (81.25%). The profile of abnormalities observed were del(6)(q24), +8(x2), del(11)(q13~q23), +12, +15(x2), del(12)(p13), -17, +21, +19, +18, del(13)(q31), del(14)(q24), del(17)(p13), +21, +4, +5, +11, t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Twelve patients exhibited normal karyotype (18.75%). All subjects presenting chromosomal abnormalities, by using G-banding analysis, were confirmed by SKY. In patients with normal cytogenetic, SKY analysis did not identified any criptic abnormality. Cells without any stimulant agent showed concordance with the cytogenetic profile obtained (FISH analysis). The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL cells in vitro than others mitogens. Cytogenetic aberrations detected by G-banding in addition to FISH analysis were heterogeneous. The limited spectrum of chromosomal abnormalities seen by FISH analysis may contribute to underestimate the prognostic value, where others abnormalities may be present in patient's karyotype. These results indicate that classical cytogenetic analysis can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis. Financial support: FAPESP (Proc. 07/52462-7). Disclosures: No relevant conflicts of interest to declare.

Notch/HES1 Implicated As a Tumor Suppressor Mechanism in Human AML in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3524-3524
Author(s):  
Leonard S Golfman ◽  
Sankaranarayanan Kannan ◽  
Mandy A Hall ◽  
Patrick A Zweidler-McKay
Keyword(s):  
Tumor Suppressor ◽  
Notch Signaling ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Blood Levels ◽  
T Cell Leukemogenesis ◽  
Proliferation Assays

Abstract Abstract 3524 Background: Although Notch signaling contributes to T cell leukemogenesis, the role of Notch in human AML is unclear. We and others have found that activation of Notch signaling inhibits AML growth and survival, e.g. a tumor suppressor like effect. However it is not known what the consequences of activating or inhibiting Notch signaling are in human AML in vivo. Approach: To determine whether Notch signaling would have growth inhibiting effects in vivo, we stably-transduced ML1 human AML cells with the constitutively-active forms of Notch1 and Notch2 (ICN1, ICN2), the common Notch target gene Hairy/Enhancer of Split 1 (HES1) or the pan-Notch inhibitor dominant-negative Mastermind-like (dnMAML) and performed in vivo competitive proliferation assays. Briefly, following transduction, each vector type were sorted to 50% GFP+ (containing the gene of interest) and GFP- (untransduced control cells). Groups of NSG mice were injected with these 50:50 mixtures of ML1 cells, and peripheral blood levels of GFP- and GFP+ cells were measured with flow cytometry for anti-human CD45 and GFP. Results: Peripheral blood engraftment of human CD45+ ML1 cells by week 5 was similar (2–5%) for ICN1, ICN2 and HES1 injected mice, but was significantly higher (23%) in dnMAML injected mice (Panel A). Similar to in vitro competitive proliferation assays, ICN1, ICN2 and HES1 all led to decreased relative numbers of GFP%+ cells, with 1%, 4% and 16% respectively (Panel B). Importantly, dnMAML had little effect on AML proliferation in vitro, however led to dramatic increases in GFP% as well as early morbidity and mortality due to increased leukemia burden, with 93% GFP+ (Panel B), demonstrating a selective advantage for dnMAML-expressing ML1 in vivo. When GFP+ (transduced) ML1 peripheral engraftment was directly compared to GFP- (parental CD45+) engraftment, the GFP- control cells had similar engraftment rates (2–5%) across groups of mice, while the GFP+ engraftment rates were significantly lower in ICN1, ICN2, and HES1 groups, but significantly higher in the dnMAML group (Panel C), demonstrating enhanced engraftment/proliferation in dnMAML-expressing cells. Conclusions: This suggests a previously unreported concept, namely that endogenous Notch ligands can inhibit human AML growth in vivo. This data supports the hypothesis that Notch behaves as a tumor suppressor in AML, and suggests the potential use of Notch agonists in human AML. Disclosures: No relevant conflicts of interest to declare.

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