scholarly journals Presence of Germline and Full-Length IgA RNA Transcripts Among Peritoneal B-1 Cells

1998 ◽  
Vol 6 (1-2) ◽  
pp. 81-87 ◽  
Author(s):  
Rick De Waard ◽  
Peter M. Dammers ◽  
James W. Tung ◽  
Aaron B. Kantor ◽  
Jennifer A. Wilshire ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Cell Lineage ◽  
Cell Subset ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Physiological Conditions ◽  
Rna Transcripts ◽  
Iga Response ◽  
B Cell Subsets

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgAin vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline CαmRNA and mature CαmRNA transcripts. Germline Cαand mature Cαtranscripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-l-cell population in CD5+B-1a cells and CD5-B-1b cells, it was shown thatin vivoexpression of germline Cαand mature Cαtranscripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.

Overexpression of the Receptor for Hyaluronan-Mediated Motility (RHAMM) Characterizes the Malignant Clone in Multiple Myeloma: Identification of Three Distinct RHAMM Variants

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1684-1696 ◽  
Author(s):  
Mary Crainie ◽  
Andrew R. Belch ◽  
Michael J. Mant ◽  
Linda M. Pilarski
Keyword(s):  
Multiple Myeloma ◽  
Crohn’S Disease ◽  
B Cells ◽  
Crohn's Disease ◽  
Plasma Cells ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Activated B Cells

The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM−48, and RHAMM−147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM−48 and RHAMM−147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM−48, and 4% were RHAMM−147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn’s disease. RHAMM−48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn’s disease. RHAMM−147 was undetectable in normal and Crohn’s disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1.2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM−48, and RHAMM−147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo–activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.

Overexpression of the Receptor for Hyaluronan-Mediated Motility (RHAMM) Characterizes the Malignant Clone in Multiple Myeloma: Identification of Three Distinct RHAMM Variants

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1684-1696 ◽  
Author(s):  
Mary Crainie ◽  
Andrew R. Belch ◽  
Michael J. Mant ◽  
Linda M. Pilarski
Keyword(s):  
Multiple Myeloma ◽  
Crohn’S Disease ◽  
B Cells ◽  
Crohn's Disease ◽  
Plasma Cells ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Activated B Cells

Abstract The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM−48, and RHAMM−147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM−48 and RHAMM−147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM−48, and 4% were RHAMM−147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn’s disease. RHAMM−48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn’s disease. RHAMM−147 was undetectable in normal and Crohn’s disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1.2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM−48, and RHAMM−147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo–activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.

Protein Signature of LMP1 Signaling in PTLDs Identifies and Mimics Inter/Perifollicular CD30+ EBV− B Blasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3251-3251
Author(s):  
Rita Shaknovich ◽  
Katia Basso ◽  
Govind Bhagat ◽  
Bachir Alobeid ◽  
Giorgio Cattoretti
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Subset ◽  
Cellular Transformation ◽  
Cell Types ◽  
Marginal Zone B Cells ◽  
Latently Infected ◽  
Protein Signature

Abstract EBV-associated B-cell Post-Transpant Lymphoproliferative Disorders (PTLDs) represent a diverse group of lesions morphologically, in clinical presentation and behaviour, ranging from early reversible lesions to monomorphic aggressive lymphomas. Polymorphic cases, which represent the focus of our analysis, contain a mixture of cells in various EBV latency stages, defined by EBNA1, EBNA2 and LMP1 immunostaining. LMP1 is a key viral protein for cellular transformation and, analogously to CD40, engages TNF Receptor Associated Proteins and activates NF-kB and NF-kB-responsive genes. We analyzed the protein signature of LMP1 in PTLDs and non-PTLD tonsils by double staining for LMP1, CD30, CD20, Pax5 and signaling molecules. A remarkably conserved set of proteins, associated with LMP1/CD40 signaling and NF-kB activation is expressed both in the EBV-infected lymphoid population in polymorphic PTLDs and in a normal B-cell subset(s) in reactive tonsils. These proteins include highly expressed CD30, JunB, nuclear cRel, TRAF-1, Bcl-XL, MUM1, CCL22 and downregulated BCL6 and CD10. We observed that EBV infection, possibly through LMP1 and LMP2A signaling, results in varioius degrees of differentiation within the neoplastic clone. EBER+ terminally differentiated mucosa-associated IRTA-1+ marginal zone B-cells and CD138+ plasma cells were identified in most cases, including control post-transplant tonsils with no overt disease. We document for the first time in situ, in-vivo evidence of EBV latently infected post-Germinal Center B cells of marginal and plasma cell types in PTLDs. Polymorphic PTLD cases represent EBV-induced expansion of B cells, mimicking CD40L-like activated Peri/Interfollicular CD30+ normal B-cells.

In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4739-4739
Author(s):  
Cristina Castilla-LLorente ◽  
Mineo Iwata ◽  
Marco Mielcarek ◽  
V. Kraig Abrams ◽  
Billanna Hwang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lymph Nodes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Blood Mononuclear Cells ◽  
Post Infusion

Abstract Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Real-Time RT-PCR Analysis of Individual Osteolineage Cells within the Hematopoietic Stem Cell Niche

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2389-2389
Author(s):  
Lev Silberstein ◽  
Masatake Osawa ◽  
Charles Lin ◽  
Peter Kharchenko ◽  
Cristina Lo Celso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Chi Squared ◽  
The Individual ◽  
Osteolineage Cells ◽  
Angiopoietin 1

Abstract Abstract 2389 Osteolineage cells (OLCs) have been shown to participate in a regulatory bone marrow microenvironment for the hematopoietic stem and progenitor cells (HSPCs) – the endosteal niche. Our previous experiments using live animal imaging have demonstrated that single transplanted HSPCs preferentially home in close proximity to the individual OLCs. We hypothesized that these HSPC-proximal cells represent a distinct subpopulation of OLCs, which is specifically involved in a non-cell autonomous regulation of HSPC quiescence and self-renewal. To test this hypothesis, we developed a novel experimental platform, which allows visualization of HSPC-OLC cell pairs in-vivo and retrieval of the individual OLCs for molecular analysis. We intravenously injected DiI labeled adult bone marrow-derived FACS-sorted Lin−Sca1+c-kit+CD34−Flk2− HSPCs into irradiated newborn collagen 2.3GFP mouse recipients; in this transgenic strain, the majority of the OLCs are labeled with green fluorescent protein (GFP). 48 hours later, we sacrificed the animals and obtained fresh unfixed sections of femoral trabecular bone. Using a combination of differential interference contrast fluorescent microscopy, in-situ enzymatic digestion and micromanipulation, we harvested individual GFP-positive OLCs located within 2 cell diameters (“niche” OLCs) or greater than 5 cell diameters (“control” OLCs) from single DiI-bright HSPCs. Following reverse transcription and cDNA amplification with 29 cycles of PCR, as per the single cell RNA-Seq protocol (Tang et al, Nature Protocols 2010), we performed real-time RT-PCR analysis of 31 samples – 15 niche cells and 16 controls - for the OLC signature genes (osteocalcin, osterix) and for the genes implicated in playing a functional role in the HSPC-OLC cell interaction (osteopontin, CXCL12, angiopoietin 1). Transcripts for GAPDH, collagen 1 and GFP served as positive controls for the amplification. As expected, all cells were positive for GFP and over 85% cells expressed collagen 1. Osteopontin and CXCL12 were expressed at a similar level and frequency in the niche and control OLCs. However, we found that angiopoietin 1 transcripts were detected exclusively in the niche OLCs (3/15 versus 0/16, p <0.05 by Chi-squared). Moreover, niche OLCs were enriched for the osterix-positive cells (7/15 versus 2/16, p <0.05 by Chi-squared) and expressed a lower level of osteocalcin, as normalized for GAPDH expression (1.13 vs. 0.97, p< 0.05 by t-test). Our results suggest that niche OLCs may have a distinct molecular signature and reside within a population of very immature OLCs, as evidenced by the osterix + osteocalcin low phenotype. Further unbiased transcriptome characterization of these cells using genome-wide RNA-Seq assay is therefore likely to provide more evidence in support of our hypothesis and reveal novel non-cell autonomous regulators of HSPC quiescence. To our knowledge, this approach represents the first attempt to define molecular heterogeneity in-vivo at a single cell level using the micro-anatomical relationship between two heterologous cell types. Disclosures: Scadden: Fate Therapeutics: Equity Ownership.

scholarly journals The Effect of Hydroxyapatite Nanocrystals on Osseointegration of Titanium Implants: AnIn VivoRabbit Study

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Karin Breding ◽  
Ryo Jimbo ◽  
Mariko Hayashi ◽  
Ying Xue ◽  
Kamal Mustafa ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Calcium Phosphates ◽  
Surface Characteristics ◽  
Healing Time ◽  
Titanium Implants ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Implant Surface ◽  
Hydroxyapatite Nanocrystals

Osseointegration is dependent on implant surface characteristics, including surface chemistry and topography. The presence of nanosized calcium phosphates on the implant surface is interesting to investigate since they affect both the nanotopography and surface chemistry, forming a bone mineral resembling surface. In this work, the osseointegration of titanium implants with and without the presence of hydroxyapatite (HA) nanocrystals has been evaluatedin vivo. The integration was examined using removal torque measurements and real-time polymerase chain reaction (RT-PCR) analysis. The study was performed using two healing time points, 3 and 12 weeks. The results showed that the torque needed to remove the implants was insignificant between the non- and HA-coated implants, both at weeks 3 and 12. The RT-PCR, however, showed significant differences for osteoblast, osteoclast, and proinflammation markers when HA nanocrystals were present.

scholarly journals A disease-causing variant of COL4A5 in a Chinese family with Alport syndrome: a case series

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jing Wu ◽  
Jun Zhang ◽  
Li Liu ◽  
Bo Zhang ◽  
Tomohiko Yamamura ◽  
...  
Keyword(s):  
Alport Syndrome ◽  
Wide Spectrum ◽  
Case Series ◽  
Pcr Analysis ◽  
Chinese Family ◽  
Compound Heterozygous ◽  
Rt Pcr ◽  
Col4a5 Gene ◽  
Minigene Assay

Abstract Background Alport syndrome (AS), which is a rare hereditary disease caused by mutations of genes including COL4A3, COL4A4 and COL4A5, has a wide spectrum of phenotypes. Most disease-causing variants of AS are located in the exons or the conservative splicing sites of these genes, while little is known about the intronic disease-causing variants. Methods A Chinese AS family was recruited in this study. All the clinical data of AS patient were collected from medical records. After pedigree analysis, the pathogenic variants were studied by the whole exome sequencing (WES). Minigene assay and in vivo RT-PCR analysis were performed to validate the functions of the variants. Results Renal biopsy showed a typical histopathology changes of AS. WES revealed compound heterozygous substitution, NM_033380 c.991–14(IVS17) A > G, in the intron 17 of the COL4A5 gene, which were confirmed by Sanger sequencing. Moreover, the variant was co-segregated with the phenotype in this family. Minigene assay in cultured cell lines showed that a splicing error was induced by this intronic variant, which further confirmed by in vivo RT-PCR analysis. Conclusion A novel intronic disease-causing variant in COL4A5 gene was identified by WES, which was the molecular pathogenic basis of AS.

scholarly journals Multiple Myeloma Includes Phenotypically Defined Subsets of Clonotypic CD20+ B Cells that Persist during Treatment with Rituximab

2008 ◽  
Vol 2 ◽  
pp. CMO.S615 ◽  
Author(s):  
Linda M. Pilarski ◽  
Eva Baigorri ◽  
Michael J. Mant ◽  
Patrick M. Pilarski ◽  
Penelope Adamson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Subset ◽  
Light Scatter ◽  
Cell Compartments ◽  
B Lineage ◽  
B Cell Subsets ◽  
Anti Cd20

Potential progenitor B cell compartments in multiple myeloma (MM) are clinically important. MM B cells and some circulating MM plasma cells express CD20, predicting their clearance by treatment with anti-CD20. Here we describe two types of clonotypic CD20+ B cell in peripheral blood of myeloma patients, identified by their expression of CD19 and CD20 epitopes, their expression of CD45RA and their light scatter properties. Thus, the circulating component of the MM clone includes at least two distinct CD19+ CD20+ B cell compartments, as well as CD138+CD20+ plasma cells. To determine whether either or both B cell subsets and the CD20+ plasma cell subset were depleted by anti-CD20 therapy, they were evaluated before, during and after treatment of patients with rituximab (anti-CD20), followed by quantifying B cell subsets over a 5 month period during and after treatment. Overall, all three types of circulating B lineage cells persist despite treatment with rituximab. The inability of rituximab to prolong survival in MM may result from this failure to deplete CD20+ B and plasma cells in MM.

Influence of Helix Length on Cleavage Efficiency of Hammerhead Ribozymes

2005 ◽  
Vol 58 (12) ◽  
pp. 851 ◽  
Author(s):  
Philip Hendry ◽  
Maxine J. McCall ◽  
Trevor J. Lockett
Keyword(s):  
Optimal Design ◽  
Base Pair ◽  
Hammerhead Ribozymes ◽  
Base Pairs ◽  
Physiological Conditions ◽  
Rna Transcripts ◽  
Cleavage Efficiency ◽  
Double Helices

The cleavage rates of RNA substrates by trans-acting, hammerhead ribozymes are controlled by interactions between helices I and II. The interactions are affected by the relative lengths of these two double helices and by unpaired nucleotides protruding beyond helix I, either in the substrate or the ribozyme strand. Maximum cleavage rates are observed for ribozyme–substrate complexes with three or more base pairs in helix II and six or less base pairs in helix I. However, for these helix combinations, rates fall sharply with unpaired nucleotides at the end of helix I. Cleavage rates by ribozymes with one or two base pairs in helix II increase as helix I is lengthened, and are unaffected by unpaired nucleotides on the end. Since miniribozymes, with one base pair in helix II, efficiently cleave long RNA transcripts under physiological conditions, they represent the optimal design for the simple hammerheads for application in vivo.

Primary Plasma Cells Expressing CD52 Are Efficiently Targeted In Vivo by Alemtuzumab.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3460-3460
Author(s):  
Carmelo Carlo-Stella ◽  
Massimo Di Nicola ◽  
Paolo Longoni ◽  
Loredana Cleris ◽  
Marco Milanesi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Plasma Cells ◽  
Therapeutic Potential ◽  
Significant Proportion ◽  
Scid Mice ◽  
Rank Test ◽  
Pcr Analysis ◽  
Significant Prolongation

Abstract Salvage therapy options for multiple myeloma (MM) patients relapsing following autologous stem cell transplantation remain limited. New treatments targeting the malignant plasma cell (PCs) are therefore needed. Alemtuzumab is a humanized monoclonal antibody to CD52 capable of destroying CD52+ cells by antibody-mediated cellular cytotoxicity and complement fixation. In order to evaluate the therapeutic potential of alemtuzumab for MM patients, we initially examined CD52 expression on primary malignant marrow PCs as well as a panel of MM continuous cell lines (RPMI-8226, KMS-11, OPM-2, U-266, LP-1, ARH-77). In addition, the anti-myeloma activity of alemtuzumab was evaluated in vivo in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. PCs were enriched from the marrow of MM patients (n = 40) according to CD138 expression. By using an immunomagnetic technique (Miltenyi Biotec, Germany, EU), highly purified CD138+ cells (median purity = 93%; median recovery = 55%) were obtained and further characterized by 3-color (CD138/CD45/CD52) flow cytometry. Expression of CD52 on CD138-enriched cells was detected in 28/40 (70%) of MM patients, with a median of 95% PCs expressing CD52. Detection of CD52 was equally evident on CD138+CD45+ and CD138+CD45− PCs (P ≥ 0.05). Similarly to what observed for primary CD138+ cells, MM cell lines showed a rather heterogeneous CD52 expression, ranging from dim (KMS-11, OPM-2) to bright positivity (LP-1, ARH-77). Both on primary PCs and MM cell lines, expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in NOD/SCID mice. Mice were inoculated intravenously with KMS-11 cells (0.5 x 105 per mice) and were treated with alemtuzumab (3 x 1 mg/mouse, subcutaneously, days 2, 5, 7). All placebo-treated mice (n = 15) died, whereas mice treated with alemtuzumab (n = 15) showed a significant prolongation of median survival (P ≤0.009 by log rank test) and 27% of them were alive and well at 120 days. No mice experienced any apparent treatment-related toxicity. According to these data, we conclude that: (1) CD52 is expressed on the plasma cells of a significant proportion of MM patients; (2) alemtuzumab has a strong antitumor activity in vivo on CD52-positive MM cell lines; (3) alemtuzumab might have therapeutic potential in a subset of MM patients.

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