scholarly journals An Exploration of the Impact of Anticentromere Antibody on Early-Stage Embryo

2017 ◽  
Vol 2017 ◽  
pp. 1-4
Author(s):  
Ying Ying ◽  
Xi Guo ◽  
Yiping Zhong ◽  
Canquan Zhou
Keyword(s):  
Early Stage ◽  
Protein A ◽  
Immunofluorescence Assay ◽  
Mouse Embryos ◽  
Anticentromere Antibody ◽  
Developmental Potential ◽  
Stage Embryo ◽  
Direct Target ◽  
The Impact

Background. Previously, we found women with positive anticentromere antibody showed impaired potential of oocyte maturation and embryo cleavage; the possible mechanism behind this phenomenon was still unknown. Objective. Thus, the present study aimed to preliminarily explore whether ACA could penetrate into the living embryos and impair their developmental potential via in vitro coculture with mouse embryos. Methods. Mouse embryos were collected and used for in vitro culture with polyclonal anticentromere protein A (CENP-A) antibody; then, immunofluorescence assay was performed to determine the penetration of antibody into embryos, and embryo development potential was observed. Results. All embryos cultured with anti-CENP-A antibody exhibited immunofluorescence on the nucleus, while none of the embryos from the control groups showed immunofluorescence. Additionally, embryos cultured with anti-CENP-A antibody experienced significant growth impairment compared with controls. Conclusion. Mouse embryos may be a direct target for ACA in vitro prior to implantation. However, the precise mechanism needs further clarification.

scholarly journals Anticentromere antibody induced by immunization with centromere protein and Freund’s complete adjuvant may interfere with mouse early-stage embryo

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hanyan Liu ◽  
Yufen Zhang ◽  
Haiying Liu ◽  
Qing Huang ◽  
Ying Ying
Keyword(s):  
Early Stage ◽  
Protein A ◽  
Immunofluorescence Assay ◽  
Immunofluorescence Test ◽  
Anticentromere Antibody ◽  
Indirect Immunofluorescence Test ◽  
Centromere Protein ◽  
Centromere Protein A ◽  
Stage Embryo ◽  
Early Stage Embryo

Abstract Background Anticentromere antibody (ACA) is a member of the antinuclear antibody spectrum (ANAs) which has been speculated to be associated with subfertility. Thus, the present study aimed to investigate the induction of ACA production and its potential interference with early-stage embryos. Methods Recombinant centromere protein-A (CENP-A) or centromere protein-B (CENP-B) and complete Freund’s adjuvant (CFA) were used to immunize mice. Serum ACA level was then evaluated by using an indirect immunofluorescence test. Immunofluorescence assay was performed to detect IgG in follicles in ovarian tissues and early-stage embryos. Results Following treatment, serum positive ACA was observed in mice treated with CENP and CFA. Furthermore, IgG were detected in follicular fluid and early-stage embryos from mice treated with CENP and CFA. Conclusions This study preliminarily indicated that ACA induced by CENP and CFA may penetrate into the living embryos of early-stage in mice.

scholarly journals Miniaturization and Automation of a Human In Vitro Blood–Brain Barrier Model for the High-Throughput Screening of Compounds in the Early Stage of Drug Discovery

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 892
Author(s):  
Elisa L. J. Moya ◽  
Elodie Vandenhaute ◽  
Eleonora Rizzi ◽  
Marie-Christine Boucau ◽  
Johan Hachani ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Blood Brain Barrier ◽  
Early Stage ◽  
Molecular Transport ◽  
Brain Barrier ◽  
Blood Brain ◽  
Cns Drugs ◽  
The Impact ◽  
The Brain

Central nervous system (CNS) diseases are one of the top causes of death worldwide. As there is a difficulty of drug penetration into the brain due to the blood–brain barrier (BBB), many CNS drugs treatments fail in clinical trials. Hence, there is a need to develop effective CNS drugs following strategies for delivery to the brain by better selecting them as early as possible during the drug discovery process. The use of in vitro BBB models has proved useful to evaluate the impact of drugs/compounds toxicity, BBB permeation rates and molecular transport mechanisms within the brain cells in academic research and early-stage drug discovery. However, these studies that require biological material (animal brain or human cells) are time-consuming and involve costly amounts of materials and plastic wastes due to the format of the models. Hence, to adapt to the high yields needed in early-stage drug discoveries for compound screenings, a patented well-established human in vitro BBB model was miniaturized and automated into a 96-well format. This replicate met all the BBB model reliability criteria to get predictive results, allowing a significant reduction in biological materials, waste and a higher screening capacity for being extensively used during early-stage drug discovery studies.

scholarly journals Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

2020 ◽  
Author(s):  
Vera A van der Weijden ◽  
Meret Schmidhauser ◽  
Mayuko Kurome ◽  
Johannes Knubben ◽  
Veronika L Flöter ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Early Stage ◽  
Developmental Competence ◽  
Molecular Signaling ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Molecular Signaling Pathways ◽  
Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

P–182 The mechanism of mouse embryo hatching and the impact of laser drilling the zona pellucida: an RNA sequencing study

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Liu ◽  
C Jones ◽  
K Coward
Keyword(s):  
Treatment Group ◽  
Mouse Embryo ◽  
Mouse Embryos ◽  
Z Score ◽  
Rna Seq ◽  
Metabolism Pathway ◽  
Hatching Process ◽  
The Impact

Abstract Study question What is the mechanism of embryo hatching? Will laser-assisted zona pellucida (ZP) drilling alter the embryonic transcriptome? Summary answer Hatching is an ATP-dependent process. Hatching is also associated with Rho-mediated signaling. Laser-assisted ZP drilling might cause alternation in embryo metabolism. What is known already Embryo hatching is a vital process for early embryo development and implantation. Animal data suggests that hatching is the result of multiple factors, such as mechanical pressure, protease activation, and the regulation of maternal secretions. However, little is known about the regulatory signaling mechanisms and the molecules involved. In addition, despite the extensive use of laser-assisted ZP drilling in the clinic, the safety profile of this technique at molecular level is very sparse. The impact of this technique on the embryonic transcriptome has not been studied systematically. Study design, size, duration Eighty mouse embryos were randomly divided into a laser ZP drilling group (n = 40) and an untreated group (n = 40). After treatment, embryos were cultured in vitro for two days. Then, hatching blastocyst (n = 8) and pre-hatching blastocyst (n = 8) from the untreated group, and the hatching blastocyst from the treatment group (n = 8) were processed for RNA sequencing (RNA-seq). Participants/materials, setting, methods Cryopreserved 8-cell stage mouse embryos (B6C3F1 × B6D2F1) were thawed, and a laser was used to drill the embryo ZP in the treatment group. Next, the treated and untreated embryos were individually cultured in vitro to the E4.5 blastocyst stage. The resulting blastocysts were lysed individually and used for subsequent cDNA library preparation and RNA-seq. Following data quality control and alignment, the RNA-seq data were processed for differentially expressed gene analysis and downstream functional analysis. Main results and the role of chance According to the RNA-seq data, 275 differentially expressed genes (DEGs) (230 up-regulated and 45 down-regulated, adjusted P < 0.05) were identified when comparing hatching and pre-hatching blastocysts in the control groups. Analysis suggested that the trophectoderm is the primary cell type involved in hatching, and revealed the potential molecules causing increased blastocyst hydrostatic pressure (Aqp3 and Cldn4). Functional enrichment analysis suggested that ATP metabolism and protein synthesis were activated in hatching blastocysts. DEGs were found to be significantly enriched in several gene ontology terms, particularly in terms of the organization of the cytoskeleton and actin polymerisation (P < 0.0001). Furthermore, according to QIAGEN ingenuity pathway analysis results, Rho signaling was implicated in blastocyst hatching (Actb, Arpc2, Cfl1, Myl6, Pfn1, Rnd3, Septin9, z-score=2.65, P < 0.0001). Moreover, the potential role of hormones (estrogen (z-score=2.24) and prolactin (z-score=2.4)) and growth factors (AGT (z-score=2.41) and FGF2 (z-score=2.213)) were implicated in the hatching process as indicated by the upstream regulator analysis. By comparing the transcriptome between laser-treated and untreated hatching blastocysts, 47 DEGs were identified (adjusted P < 0.05) following laser-assisted ZP drilling. These genes were enriched in metabolism-related pathways (P < 0.05), including the lipid metabolism pathway (Mvd, Mvk, Aacs, Gsk3a, Pik3c2a, Aldh9a1) and the xenobiotic metabolism pathway (Aldh18a1, Aldh9a1, Keap1, and Pik3c2a). Limitations, reasons for caution Findings in mouse embryos may not be fully representative of human embryos. Furthermore, the mechanism of hatching revealed here might only reflect the hatching process of embryos in vitro. Further studies are now necessary to confirm these findings in different conditions and species to determine their clinical significance. Wider implications of the findings: Our study profiled the mouse embryo transcriptome during in vitro hatching, identified potential key genes and mechanisms for future study. In addition, for the first time, we revealed the impact of laser-assisted ZP drilling on the transcriptome, this may help us to assess and improve the existing technique. Trial registration number Not applicable

185 EMBRYONIC LOSS IN PIGS ASSOCIATED WITH OVIDUCT TRANSPLANTATION OF EARLY-STAGE EMBRYOS WITH DAMAGES IN THE ZONA PELLUCIDA

2006 ◽  
Vol 18 (2) ◽  
pp. 200
Author(s):  
S. Ueno ◽  
M. Kurome ◽  
R. Tomii ◽  
K. Hiruma ◽  
N. Maeda ◽  
...  
Keyword(s):  
Immune Response ◽  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Early Stage ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Embryo Recovery ◽  
Embryonic Loss ◽  
The Impact

It is assumed that if porcine early-stage embryos with damages in their zonae pellucidae are transplanted to the recipient's oviduct, they may suffer from mechanical and immunological stresses by oviduct contraction and the recipient's immune response. This study aimed to examine the impact of zona pellucida damages, which might arise during nuclear transfer and intra cytoplasmic sperm injection (ICSI), on the development and survival of transplanted embryos. Cumulus-oocyte complexes were collected from ovaries obtained at a local slaughterhouse and matured in vitro in NCSU23 to prepare MII-stage oocytes. The zonae pellucidae of these oocytes were either penetrated with 8- to 10-�m square-ended microinjection pipettes or incised with 35- to 40-�m beveled enucleation pipettes. Intact oocytes were used as controls. The oocytes were electroactivated to induce parthenogenesis and transplanted to the oviducts of estrus-synchronized recipient gilts (estrus-synchronized with 1000 IU eCG and 1500 IU hCG). After 5 to 7 days, the recipient uteri were flushed with PBS supplemented with 1% fetal bovine serum (FBS) to collect embryos, and their development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-penetrated, 129 zona-incised, and 57 intact embryos were transplanted to four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (59.0 to 81.8%). However, the zona-penetrated and zona-incised embryos showed inconsistent development and survival compared with controls; the development and survival rate were 92.6% (25/27) to 96.7% (29/30) and 77.8% (21/27) to 96.7% (29/30) in control embryos, respectively, whereas those of zona-penetrated embryos were 57.1% (28/49) to 95.7% (22/23) and 8.2% (4/49) to 78.3% (18/30), and those of zona-incised embryos were 47.6% (30/63) to 92.3% (36/39) and 23.8% (15/63) to 92.3% (22/23), respectively. Large foci of cells that appeared to be macrophage giant cells were observed at the surface or inside of the degenerated zona-damaged embryos. These results indicate that the recipient's immune response may impair development after transplantation of the embryo to the oviduct, when there is damage in the zona pellucida. This may be one of the factors attributable to the reduced efficiency of live progeny production by ICSI and nuclear transfer. This work was supported by PROBRAIN.

scholarly journals Introduction of the mec Element (Methicillin Resistance) into Staphylococcus aureus Alters In Vitro Functional Activities of Fibrinogen and Fibronectin Adhesins

1998 ◽  
Vol 42 (3) ◽  
pp. 564-570 ◽  
Author(s):  
Pierre E. Vaudaux ◽  
Vincenza Monzillo ◽  
Patrice Francois ◽  
Daniel P. Lew ◽  
Tim J. Foster ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Bacterial Colonization ◽  
Protein A ◽  
Methicillin Resistant ◽  
Functional Activities ◽  
Extracellular Matrix Components ◽  
Resistant Strains ◽  
The Impact

ABSTRACT Some methicillin-resistant strains of Staphylococcus aureus are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (mec) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intactmec element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the femA,femB, or femC gene, known to decrease expression of methicillin resistance in S. aureus. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a mec element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the mecA gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of themec element into genetically defined strains of S. aureus impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the mecA gene.

scholarly journals MgrA Governs Adherence, Host Cell Interaction, and Virulence in a Murine Model of Bacteremia Due to Staphylococcus aureus

2019 ◽  
Vol 220 (6) ◽  
pp. 1019-1028 ◽  
Author(s):  
Liang Li ◽  
Genzhu Wang ◽  
Ambrose Cheung ◽  
Wessam Abdelhady ◽  
Kati Seidl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Virulence Gene ◽  
Toxin Production ◽  
Cell Interaction ◽  
Host Pathogen Interactions ◽  
Host Pathogen ◽  
Direct Binding ◽  
The Impact

AbstractBackgroundMgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains.MethodsIn vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model.ResultsmgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed.ConclusionsThese results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections.

scholarly journals Paraquat Preferentially Induces Apoptosis of Late Stage Effector Lymphocyte and Impairs Memory Immune Response in Mice

2019 ◽  
Vol 16 (11) ◽  
pp. 2060 ◽  
Author(s):  
Yiming Shao ◽  
Yifan Zhao ◽  
Tingting Zhu ◽  
Fen Zhang ◽  
Xiuli Chang ◽  
...  
Keyword(s):  
Immune Response ◽  
Late Stage ◽  
Early Stage ◽  
Keyhole Limpet Hemocyanin ◽  
Primary Immune Response ◽  
Secondary Immune Response ◽  
Effector Cells ◽  
Memory Immune Response ◽  
The Impact

Paraquat (PQ) is a toxic non-selective herbicide. To date, the effect of PQ on memory immune response is still unknown. We investigated the impact of PQ on memory immune response. Adult C57BL/6 mice were subcutaneously injected with 2 mg/kg PQ, 20 mg/kg PQ or vehicle control every three days for two weeks. A single injection of keyhole limpet hemocyanin (KLH) at day four after the initial PQ treatment was used to induce a primary immune response; a second KLH challenge was performed at three months post the first KLH immunization to induce a secondary immune response. In steady state, treatment with 20 mg/kg PQ reduced the level of serum total IgG, but not that of IgM; treatment with 20 mg/kg PQ decreased the number of effector and memory lymphocytes, but not naïve or inactivated lymphocytes. During the primary immune response to KLH, treatment with 20 mg/kg PQ did not influence the proliferation of lymphocytes or expression of co-stimulatory molecules. Instead, treatment with 20 mg/kg PQ increased the apoptosis of lymphocytes at late stage, but not early stage of the primary immune response. During the secondary immune response to KLH, treatment with 20 mg/kg PQ reduced the serum anti-KLH IgG and KLH-responsive CD4 T cells and B cells. Moreover, effector or activated lymphocytes were more sensitive to PQ-induced apoptosis in vitro. Treatment with 2 mg/kg PQ did not impact memory immune response to KLH. Thus, treatment with 20 mg/kg PQ increased apoptosis of late stage effector cells to yield less memory cells and thereafter impair memory immune response, providing a novel understanding of the immunotoxicity of PQ.

Putrescine supplementation during in vitro maturation of aged mouse oocytes improves the quality of blastocysts

2017 ◽  
Vol 29 (7) ◽  
pp. 1392 ◽  
Author(s):  
Dandan Liu ◽  
Guolong Mo ◽  
Yong Tao ◽  
Hongmei Wang ◽  
X. Johné Liu
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
In Vitro Fertilisation ◽  
Aged Mouse ◽  
Developmental Potential ◽  
The Impact

Mouse ovaries exhibit a peri-ovulatory rise of ornithine decarboxylase and its product putrescine concurrent with oocyte maturation. Older mice exhibit a deficiency of both the enzyme and putrescine. Peri-ovulatory putrescine supplementation in drinking water increases ovarian putrescine levels, reduces embryo resorption and increases live pups in older mice. However, it is unknown if putrescine acts in the ovaries to improve oocyte maturation. This study examined the impact of putrescine supplementation during oocyte in vitro maturation (IVM) on the developmental potential of aged oocytes. Cumulus–oocyte complexes from 9–12-month-old C57BL/6 mice were subjected to IVM with or without 0.5 mM putrescine, followed by in vitro fertilisation and culture to the blastocyst stage. Putrescine supplementation during IVM did not influence the proportion of oocyte maturation, fertilisation or blastocyst formation, but significantly increased blastocyst cell numbers (44.5 ± 1.9, compared with 36.5 ± 1.9 for control; P = 0.003). The putrescine group also had a significantly higher proportion of blastocysts with top-grade morphology (42.9%, compared with 26.1% for control; P = 0.041) and a greater proportion with octamer-binding transcription factor 4 (OCT4)-positive inner cell mass (38.3%, compared with 19.8% for control; P = 0.005). Therefore, putrescine supplementation during IVM improves egg quality of aged mice, providing proof of principle for possible application in human IVM procedures for older infertile women.

Tumor Cells Resistant to Alemtuzumab Complement Mediated Cytotoxicity in Patients with High Risk Previously Untreated Early Stage CLL: A Possible Mechanism of Treatment Failure.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2973-2973
Author(s):  
Clive S. Zent ◽  
Nancy D. Bone ◽  
Susan M. Geyer ◽  
Neil E. Kay
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
High Risk ◽  
B Cell ◽  
Cell Clone ◽  
Early Stage ◽  
Cell Membrane Permeability ◽  
Viable Cells ◽  
The Impact

Abstract The monoclonal antibodies (MoAb) alemtuzumab and rituximab have proven efficacy in the treatment of CLL. In addition, alemtuzumab is effective in patients with defective p53 function responding poorly to purine analogue therapy. The action of both MoAb is not completely understood. Proposed mechanisms include complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and direct induction of apoptosis of CLL B cells. We have done correlative studies on CLL B cells from patients enrolled in a trial of alemtuzumab and rituximab in “high risk” early stage previously untreated CLL to determine: 1. Role of apoptosis induction and CDC in each MoAb and 2. If the addition of rituximab to alemtuzumab increases their in vitro cytotoxicity. Patients and Methods: Patients with early stage, previously untreated, high risk CLL are treated with subcutaneous alemtuzumab (dose escalation over 3 days then 30 mg Mon-Wed-Fri for 4 weeks) and rituximab (375 mg/m2/dose weekly from day 8 x 4 doses). High risk disease was defined as one or more of the following features of the CLL B cell clone: (1) 17p13−; (2) 11q22−; (3) unmutated IgVH (< 2%) and either CD38+ or ZAP-70+. Blood B lymphocytes collected prior to the start of therapy were tested for response to MoAb in vitro. Cells were cultured at 2 x 106/ml in AIM-V medium using standard conditions. Alemtuzumab and rituximab were used at 20 μg/ml and complement as 10% of 40 CH50 units/ml human serum. The impact of the MoAb was measured by counting viable cells (trypan blue negative) and measuring early apoptosis (annexin V) and cell death (cell membrane permeability to propidium iodide) using flow cytometry at 1 hour, and then daily for 3 days. Results: Treatment caused rapid resolution of lymphocytosis in all 7 patients and 3 patients were negative for circulating CLL cells using a highly sensitive 3 color flow cytometry (CD5+/CD19+/CD79b-) after therapy. All patients had a clinical response (2 CR, 5 PR). Alemtuzumab and complement were rapidly cytotoxic to most CLL cells. Mean cell viability was 39% (sd: 8%) after 1 hour of incubation. Cytotoxicity was similar in all samples irrespective of FISH defects, IgVH mutation status, and in vitro resistance to F-ara-A (n = 3). Alemtuzumab was inactive in the absence of complement for all samples. Rituximab alone and together with complement did not induce cytotoxicity or apoptosis. However, the addition of rituximab to alemtuzumab and complement did increase CDC where the number of viable cells was significantly lower at 1, 24, 48, and 72 hours incubation (p = 0.075, 0.047, 0.031, 0.027, respectively, for pairwise comparisons). CLL cells surviving alemtuzumab CDC subsequently had a lower level of apoptosis than control cells, implying a selection for resistant cells. Alemtuzumab CDC on this residual population was not increased at higher concentrations of alemtuzumab or complement. This mechanism of CDC resistance is currently under investigation. Conclusion: These data suggest that alemtuzumab CDC is an important mechanism of action in patients with CLL. However, alemtuzumab CDC kills only about 61% of CLL cells in vitro, and the surviving cells are more resistant to spontaneous apoptosis. This suggests that cells that survive alemtuzimab CDC contribute to disease progression or relapse. We intend to elucidate the mechanism of this resistance using our in vitro model with the hope that treatment strategies can be deployed to remove this residual CLL B cell clone.

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