Blood Donor Leukocyte Reduction Filters as a Source of Human B Lymphocytes

Weitkamp, Jörn-Hendrik; Crowe, James E.

doi:10.2144/01313bm01

Cited by 21 publications

(20 citation statements)

References 1 publication

(1 reference statement)

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“…Normal peripheral mononuclear cells were obtained using leukocyte reduction filters from the Red Cross. 18 Peripheral blood mononuclear cells (PBMC) were isolated utilizing density gradient centrifugation (Ficoll-Paque Plus, Pharmacia Biotech, Piscataway, NJ, USA), washed in phosphate-buffered saline (PBS), and incubated at 10 7 cells/ml. The 697 lymphoblastoid cell lines stably transfected either with vector alone or vector containing the Bcl-2 gene have been described.…”

Section: Cell Culturementioning

confidence: 99%

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells

Lucas

Davis²,

Parthun³

et al. 2004

Leukemia

121

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MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC 50 that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.

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Section: Cell Culturementioning

confidence: 99%

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells

Lucas

Davis²,

Parthun³

et al. 2004

Leukemia

121

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“…Blood was obtained from two adult patients (donors 5 and 6) who were naturally exposed to RV and reported symptoms of RV disease within 1 mo prior. Blood for RV-specific B cell analysis from eight healthy blood donors (donors [7][8][9][10][11][12][13][14] was collected from leukocyte reduction filters as described (13). For randomly selected B cell controls, we obtained blood cells from leukocyte reduction filters from three healthy blood donors (donors [15][16][17] by the same method.…”

Section: Generation Of Human Rv-specific B Cell Clonesmentioning

confidence: 99%

“…The recovery of RV VP7-specific V H and V L segments following expansion was four to five times more efficient in recently RV-exposed adults than in healthy adult blood donors or RV-infected infants. We analyzed a total of 30 RV-specific V H and 39 RV-specific V L segments from recently RV-exposed adults (donors 5 and 6) and 62 RV-specific V H and 46 RV-specific V L segments from healthy adult blood donors (donors [7][8][9][10][11][12][13][14]. For the control group (donors 15-17), a total of 83 V H and 46 V L segments from randomly selected (not virus-specific) B cell clones was analyzed.…”

Section: Adults (Donors 5-17)mentioning

confidence: 99%

Infant and Adult Human B Cell Responses to Rotavirus Share Common Immunodominant Variable Gene Repertoires

Weitkamp

Kallewaard

Kusuhara

et al. 2003

The Journal of Immunology

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Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (VH and VL) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the VH1 and VH4 families, for example, VH1–46, VH4–31, and VH4–61. This gene segment bias differed markedly from the VH3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant VH segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2–11 mo and those of adults were highly related when compared by VH, D, JH, VL, and JL segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.

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“…Peripheral blood mononuclear cells (PBMC) were isolated from whole blood immediately following donation using ficoll density gradient centrifugation (Ficoll-Paque Plus, Amersham Biosciences, Piscataway, NJ, USA) and depleted of non-B cells using the Miltenyi Biotec (Auburn, CA, USA) B-Cell Isolation Kit. T cells were obtained from random blood donors using a technique to isolate mononuclear cells from leukocyte reduction filters 29 with subsequent negative selection of T cells using a pan T-cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). The lymphocytic concentrate was greater than 90% CD3 positive cells as determined by FACS analysis.…”

Section: Methodsmentioning

confidence: 99%

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism

et al. 2005

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Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cellmembrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab þ anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

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Blood Donor Leukocyte Reduction Filters as a Source of Human B Lymphocytes

Cited by 21 publications

References 1 publication

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells

Infant and Adult Human B Cell Responses to Rotavirus Share Common Immunodominant Variable Gene Repertoires

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism

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