Identification Of Cell Surface Antigens Research Articles

Platelet rich plasma enhanced neuro-regeneration of human dental pulp stem cells in vitro and in rat spinal cord.

BackgroundHuman dental pulp stem cells (hDPSCs) exhibit excellent differentiation potential and are capable of differentiating into several different cellular phenotypes, including neurons. Platelet-rich plasma (PRP) contains numerous growth factors that can stimulate stem cell differentiation. In this study, we investigated the potential stimulatory effects of PRP on neurogenic differentiation and anti-apoptosis of hDPSCs in injured spinal cords.MethodsThe unipotential differentiation capacity of hDPSCs was analyzed by cell surface antigen identification and cell cycle analysis. A spinal cord injury rat model composed of 40 Sprague-Dawley (SD) rats was used to facilitate an in vivo study. Rats were divided into four groups: a double-treatment group (receiving both neurogenic-induced hDPSCs and PRP), two single-treatment groups (receiving neurogenic-induced hDPSCs or PRP) and a sham group (receiving normal saline). The Basso, Beattie, Bresnahan Locomotor Rating Scale was subsequently used to evaluate the motor function of the spinal cord. Cell viability and differentiation of hDPSCs in the damaged spinal cords were analyzed and apoptosis of neural cells was evaluated using the terminal uridine nucleotide end labeling (TUNEL) assay.ResultsGrowth pattern, cell surface marker and cell cycle analyses revealed that hDPSCs have a high degree of multi-directional differentiation potential and can be induced into neurons in vitro. In the rat spinal cord injury model, double-treatment with hDPSC/PRP or single treatment with hDPSCs or PRP significantly improved motor function compared with the sham group (P<0.05). Apoptosis of neural cells was observed to be significantly higher in the sham group compared to any of the treatment groups. Double-treatment with hDPSCs and PRP resulted in the lowest apoptotic rate among the groups analyzed.ConclusionshDPSCs exhibit differentiation potential and are capable of transforming into neural cells both in vitro and in vivo. Significantly increased inhibition of neuronal apoptosis and improved motor function recovery of the spinal cord were observed following double-treatment with hDPSCs and PRP compared with the single-treatment groups.

3058 – SURFACE PROTEOMICS REVEALS NOVEL POTENTIAL AML ANTIGENS FOR IMMUNOTHERAPEUTIC TARGETING

Compared to solid tumors, antibody-based immunotherapy is underexploited in acute myeloid leukemia (AML), likely due to the limited availability of targetable surface antigens specifically expressed on AML cells. While targeted immunotherapies hold great promise, their development inexorably depends on the identification of cell surface antigens selectively expressed by cancer cells. The identification of such antigens has been challenging due to the characteristic hydrophobicity of plasma membrane proteins. Another level of complexity is brought by the fact that AML is a heterogeneous disease comprised of different subgroups with associated prognosis ranging from favorable to adverse. In order to identify novel AML antigens to be targeted using immunotherapeutic approaches, we have analysed the surface proteome of a collection of 100 primary human AML specimens, enriched in poor prognosis AML samples. These analyses revealed that AML subgroups have distinct surfaceome signatures. We also confirmed the expression of known AML markers at the surface of specific AML subgroups. Our search for novel AML antigens focused on four poor prognosis AML subgroups: MLL-rearranged, complex karyotype, RUNX1-mutated and normal karyotype with NPM1, DNMT3A and FLT3 (ITD) mutations. We identified proteins selectively expressed at the surface of AML cells from these subgroups. Analysis of the whole cohort of specimens also identified known and novel pan AML antigens. Single cell RNA-sequencing analysis of aforementioned subgroup-specific and pan AML antigens revealed that many of these are expressed by the vast majority of blast populations within primary AML specimens, including most primitive ones. Overall, these results suggest that studying the surface proteome of AML cells has the potential to reveal promising AML antigens to be targeted using immunotherapeutic approaches.

Pre-Clinical Evaluation of B7-H3-Specific Chimeric Antigen Receptor T-Cells for the Treatment of Acute Myeloid Leukemia

▪Background: The development of safe and effective chimeric antigen receptor T-cell (CAR-T) therapy for acute myeloid leukemia (AML) remains an elusive goal. Whereas CD19-directed CAR-T therapies have shown great promise for the treatment of B-cell malignancies, the identification of AML-associated surface antigens that can be safely and effectively targeted by CAR T-cells has remained a challenge. Because most AML-associated surface antigens are also expressed on normal myeloid progenitors, the potential for unacceptable hematopoietic toxicity has been a major limitation. The identification of cell surface antigens that are absent on all normal myeloid progenitors and yet expressed on all subtypes of AML is not likely. On the other hand, it seems plausible that some antigens not detected on early myeloid lineage cells may be preferentially overexpressed in certain AML subtypes. We have identified B7-H3 as one such candidate. B7-H3 (B7-homolog 3, or CD276) is a coreceptor belonging to the B7 family of immune checkpoint molecules. B7-H3 protein expression in normal tissues is largely restricted to certain antigen-presenting cells. In multiple human cancers, however, B7-H3 protein is significantly overexpressed. This includes a substantial subset of AML, and B7-H3 expression appears to be higher in AML with a monocytic immunophenotype. Furthermore, B7-H3 on tumor cells, and on myeloid-derived suppressor cells in the tumor microenvironment, is likely to play an immunosuppressive role, and may drive immune escape in multiple cancer types. This suggests that targeting B7-H3 could also enhance anti-tumor adaptive immune responses. We therefore hypothesized that B7-H3-specific CAR-Ts (B7-H3.CARs) could be effective in eliminating B7-H3-expressing AML cells and would not cause unacceptable hematopoietic toxicity.Methods and Results: We obtained bone marrow aspirates from patients with monocytic/myelomonocytic AML (n=10) and demonstrated surface expression of B7-H3 on a median of 62.5% (range 27.8 to 98.5) of primary AML blasts. We also showed that B7-H3 is highly expressed on monocytic/myelomonocytic AML cell lines (THP1, U937, OCI-AML2, OCI-AML3), and that B7-H3 expression compares favorably to that of other previously identified candidate antigens for AML-directed CAR-T therapy. Next, we generated B7-H3.CARs via retroviral transduction of CD3/CD28-activated T-cells, followed by expansion in vitro with interleukin- (IL) 7 and IL-15. When B7-H3.CARs (n=3-5 donors) were cocultured with B7-H3-positive AML cell lines (listed above) and with primary AML blasts (n=10 patients), B7-H3.CARs proliferated, released high amounts of IL-2 and interferon-γ, and demonstrated efficient B7-H3-specific cytotoxicity. Autologous B7-H3.CARs also demonstrated significant cytotoxicity against primary AML blasts (n=4). Additionally, B7-H3.CARs controlled tumor cell proliferation and prolonged survival in xenograft mouse models of disseminated AML using OCI-AML2 (p=0.0025, n=5 mice per group) and THP1 (p<0.0001, n=10 mice per group).Next, we showed that B7-H3 is not significantly expressed on hematopoietic stem cells or myeloid progenitor cell populations in normal human bone marrow samples (n=2). We also evaluated the effects of B7-H3.CARs (n=4 donors) on normal hematopoietic stem cells via in vitro colony formation assays using umbilical cord-blood (n=4 donors) derived CD34+ cells, and showed that B7-H3.CARs did not significantly inhibit the formation of myeloid progenitor colonies. We then showed in a humanized mouse model (using fetal liver-derived hematopoietic stem cells) that B7-H3.CARs did not lead to significant reductions in the populations of circulating CD45/CD14-positive or CD45/CD33-positive cells.Conclusions: B7-H3 is expressed on a significant proportion of AML blasts from patients with monocytic AML. Adoptive transfer of B7-H3.CARs could be an effective treatment option for patients with B7-H3-positive AML, since i) we have previously demonstrated limited expression of B7-H3 in normal tissues, and ii) the present results show that B7-H3.CARs are unlikely to cause significant hematopoietic toxicity. Given variable expression of B7-H3 in AML, however, it may be necessary to develop a dual-targeting approach, combining B7-H3 with a second target AML-associated surface antigen. DisclosuresDu:N/A: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor. Ferrone:N/A: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor. Dotti:University of North Carolina: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor.

Abstract 2211: Characterization of monoclonal antibodies specific for HPV-positive head and neck cancer

Abstract HPV-driven head and neck squamous cell carcinoma (HNSCC) are among the fastest growing cancers. Although, HPV-HNSCC patients have an overall favorable prognosis, a significant number of patients relapse post-treatment, and currently there is no specific therapeutic approach targeting the unique biology of HPV-driven HNSCC. Our ongoing efforts to target HPV-HNSCC focus on the identification of cell surface antigens that are upregulated by HPV infection. We have developed an "antigen-agnostic" approach for generating HPV-HNSCC-targeting monoclonal antibodies for cancer diagnosis and treatment that does not rely on prior identification of target antigens. We used HPV-HNSCC membrane fractions to immunize recipient mice and generated HPV-specific hybridomas. We then screened five thousand hybridoma colonies by flow cytometry to test the specificity of binding to HPV-positive cancer cell lines (2HNSCC and 2 Cervical Cancer) and HPV-negative cancer cell lines (4 HNSCC and 1 CC). After primary screening, we narrowed down to forty-four clones; among these hybridoma clones, 6D8 and 6B3 bound preferentially to HPV-positive cancer cell lines. The binding targets of 6D8 and 6B3 were identified by immunoprecipitation and mass spectrometry; their targets are integrin alpha6 (ITGA6) and tissue factor (F3) respectively. Future work will validate the biological function of these mAbs in in vitro and in vivo models, and continue identifying more binding target of mAbs. We propose mAbs specifically targeting membrane-expressed antigens on HPV-related cancer cells as a potential approach for early diagnosis and targeted therapy. Citation Format: Hsuan-Chen Liu, Falguni Parikh, Thomas Kraus, Thomas Moran, Andrew Sikora. Characterization of monoclonal antibodies specific for HPV-positive head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2017-2211

Abstract 3886: Clec12a: A new AML stem cell-associated antigen

Abstract Introduction: Acute myeloid leukemias (AML), a heterogeneous and complex group of diseases, is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs), which are sustained by self-renewing and responsible for the propagation of leukemic blasts (LBs). Monoclonal antibodies have emerged as effective targeted therapies for the treatment of human malignancies and their mechanisms of action are able to deliver the therapeutic effects with minimal toxicity. Aim: The challenge is the identification of cell surface antigens which could be preferentially expressed on AML LSC compared with normal hematopoietics stem cells and that could be helpful to target therapies. Materials and methods: On 16 AML patients (pts) at diagnosis (peripheral blood of 12 pts and bone marrow of 4 pts) myeloblast leukemic cells were identified using a FACSCanto flow cytometer (Becton Dickinson), based on low expression of CD45 and low side scatter (SSC) properties (CD45 low/SSC low). On these subpopulation we used an antibody panel against cell surface antigens, for the detection of immature markers and potential leukemia-associated antigens: CD34, CD38, CD90, CLEC12A (C-type lectin domain family 12 member A), CD44, CD99, TIM-3 (T cell immunoglobulin mucin-3), CD32, CD133, CD74, CD47, CD58, CD25, CD22, CD96. Results:. The proportion of LBs was positively correlated with CD45 low/SSC low (median 51,8 %). This gated population of LBs were positive for CD34 (median 44,65%), CD38 (median 18,75%), CD90 (median 0,90%), CLEC12A (median 93,7%), CD44 (median 99,9%), CD99 (median 96,6%), TIM-3 (median 84,4%), CD32 (median 14%), CD123 (29,15%), CD133 (median (8,95%), CD58 (median 97,5%), CD47 (median 99,9%), CD74 (median 3,2%), CD25 (median 0,6%), CD96 (median 87%), CD22 (median 0,55%). Conclusion: The expression of CD34 and CD38 antigens is heterogeneous in LBs. In particular we found that the 50% of patients were CD34+ and 50% were CD38+. CLEC12A, CD44, CD99, TIM-3, CD58, CD47 and CD96 were highly expresses in LCSs. CD90, CD32, CD123, CD133,CD74, CD25 and CD22 were low. Interestingly we identified that the expression of CLEC12A distinguished two different populations: the CLEC12Ahigh cells correlated with the blast cells CD45low/SSClow; on the other hand, CLEC12Alow cells could be compared with CD45high/SSChigh population, representing normal hematopoietic cells. In conclusion, this marker is a good candidate to target therapies against leukemic stem cells. However further studies with an higher number of patients must be carried out to confirm that CLEC12A is an appropriate antigen for a new monoclonal antibody-based therapy. Acknowledgements: European LeukemiaNet, AIL, AIRC, PRIN 2010-2011, FP7 NGS-PTL project. Citation Format: Viviana Guadagnuolo, Enrica Imbrogno, Andrea Ghelli Luserna di Rorà, Antonella Padella, Giorgia Simonetti, Emanuela Ottaviani, Cristina Papayannidis, Ilaria Iacobucci, Anna Ferrari, Simona Soverini, Giovanni Martinelli. Clec12a: A new AML stem cell-associated antigen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2014-3886

Foxc2 regulates osteogenesis and angiogenesis of bone marrow mesenchymal stem cells

BackgroundThe Forkhead/Fox transcription factor Foxc2 is a critical regulator of osteogenesis and angiogenesis of cells. Bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and fibroblasts. The present study investigates the role of Foxc2 overexpression in osteogenesis and angiogenesis of BMSCs in vitro.MethodsBMSCs were isolated from SD rat femurs and tibias, and characterized by cell surface antigen identification and osteoblasts and adipocytes differentiation. The cells were transfected with lentiviral Foxc2 (Lv-Foxc2) or green fluorescent protein (Lv-GFP). Seventy hours later, Foxc2 expression was observed using real time-PCR and Western blot. The transfected cells were stained with Alizarin red S or alkaline phosphatase (ALP) after osteogenic induction. Meanwhile, the expression levels of osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF) and platelet-derived growth factor-β (PDGF-β) were measured by real time-PCR, Western blot and immunostaining.ResultsResults of cell characterization showed that the cells were positive to CD44 (99.56%) and negative to CD34 (0.44%), and could differentiate into osteoblasts and adipocytes. Foxc2 overexpression not only increased the numbers of mineralized nodes and ALP activity, but also enhanced the expressions of Runx2, OCN, VEGF and PDGF-β in transfected BMSCs after osteogenic induction. The effects of Foxc2 on osteogenesis and angiogenesis were significantly different between Lv-Foxc2 transfected BMSCs and Lv-GFP transfected BMSCs (P<0.05). In addition, the MAPK-specific inhibitors, PD98059 and LY294002, blocked the Foxc2-induced regulation of BMSC differentiation.ConclusionsFoxc2 gene is successfully transfected into BMSCs with stable and high expression. The overexpression of Foxc2 acts on BMSCs to stimulate osteogenesis and angiogenesis. The effect of Foxc2 on angiogenesis of the cells is mediated via activating PI3K and ERK.

Purification and identification of cell surface antigens using lamprey monoclonal antibodies

Variable lymphocyte receptor (VLR) B antibodies of the evolutionary distant sea lamprey are structurally distinct from conventional mammalian antibodies. The different protein architecture and large evolutionary distance of jawless vertebrates suggest that VLR antibodies may represent promising tools for biomarker discovery. Here we report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. We demonstrate that despite predicted low affinity of individual VLR antigen binding units to the antigen, the high avidity resulting from decameric assembly of secreted VLR antibodies allows for efficient antigen capture and subsequent identification by mass spectometry. We show that VLR antibodies detect their antigens with high specificity and can be used in various standard laboratory application techniques. The lamprey antibodies are novel reagents that can complement conventional monoclonal antibodies in multiple scientific research disciplines.

Increased Integration of Transplanted CD73-Positive Photoreceptor Precursors into Adult Mouse Retina

PURPOSE. Retinal degeneration initiated by loss of photoreceptors is the prevalent cause of visual impairment and blindness in industrialized countries. Transplantation of photoreceptor cells represents a possible replacement strategy. This study determined that identification of cell surface antigens can assist in enriching photoreceptor precursors for transplantation. METHODS. The expression profile of rod photoreceptors at postnatal day 4 was investigated by microarray analysis to identify photoreceptor-specific cell surface antigens. For enrichment of transplantable photoreceptors, neonatal retinas from rod photoreceptor-specific reporter mice were dissociated, and the rods were purified by magnetic associated cell sorting (MACS) with CD73 antibodies. MAC-sorted cell fractions were transplanted into the subretinal space of adult wild-type mice. The number of rod photoreceptors contained in unsorted, CD73-negative, and CD73-positive cell fractions were quantified in vitro and after grafting in vivo. RESULTS. Microarray analysis revealed that CD73 is a marker for rod photoreceptors. CD73-based MACS resulted in enrichment of rods to 87%. Furthermore, in comparison with unsorted cell fractions, CD73-positive MAC-sorted cells showed an approximately three-fold increase in the number of integrated, outer segment-forming photoreceptors after transplantation. CONCLUSIONS. CD73-based MACS is a reliable method for the enrichment of integrating photoreceptors. Purification via cell surface markers represents a new tool for the separation of transplantable photoreceptor precursors from a heterogeneous cell population, avoiding the need of reporter gene expression in target cells.

E0173 Isolation culture and identification of rat MSC

AimsTo investigate different ways of isolating and culturing rat MSC and different serum concentrations of medium for the best selection.Materials and methodsDirect adherence and density gradient centrifugation methods are used...

Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro

Objective VEGF and BMP play important roles in angiogenesis and osteogenesis. Combining these two factors may be a promising therapeutic strategy for avascular necrosis of the femoral head (ANFH). Methods Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and purified by density gradient centrifugation combined with attachment culture methods. The purity and characteristics of the BMSCs were detected by cell surface antigen identification. The best MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting, and cell viability was determined by MTT assay. Expression of the genes of interest was detected by GFP gene expression, RT-PCR assay, and ELISA assay. The biological activities of VEGF and BMP were detected by angiogenic and osteogenic assays. Results The best MOI of BMSCs transfected with rAAV was 5 × 10 4 v.g./cell. Cell growth curves showed vigorous cell viability. Expressions of the GFP, VEGF165, and BMP 7 genes were detected 1 day post-transfection and peaked 14 days post-transfection. Expression of the genes of interest was sustained over 1 month. VEGF and BMP proteins secreted from BMSCs transfected with rAAV-hVEGF 165-IRES-hBMP 7 enhanced angiogenesis and osteogenesis in vitro. Conclusion Recombinant adeno-associated viral vectors co-expressing the hVEGF 165 and hBMP 7 genes showed efficient gene expression ability. The VEGF 165 and BMP 7 proteins expressed from the vector have efficient biological activity in vitro.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

BackgroundAvailable bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.Methods and FindingsWe applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.ConclusionsThe results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Stem cells in human breast cancer.

Increasing data support cancer as a stem cell-based disease. Cancer stem cells (CSCs) have been found in different human cancers, and recent evidence indicates that breast cancer originates from and is maintained by its own CSCs, as well as the normal mammary gland. Mammary stem cells and breast CSCs have been identified and purified in in vitro culture systems, transplantation assays and/or by cell surface antigen identification. Cell surface markers enable the functional isolation of stem cells that can initiate and propagate tumorigenesis in mammary gland. These observations have dramatic biological and clinical significance due to increasing evidence suggesting that the recurrence of human cancer and treatment failure may reflect the intrinsic quiescence and drug resistance of CSCs. Thus, the CSC hypothesis provides fundamental implications for understanding breast carcinogenesis and for developing new strategies for breast cancer prevention and therapy for advanced disease. Further strategies to isolate breast CSCs, to find additional trustworthy surface markers, and to compare gene expression pathways profiles with their normal stem cells counterparts are necessary to more accurately define putative breast cell-lineage markers for the different cell types present in the mature mammary gland and to identify potential therapeutical targets in breast cancer. This review discusses the current knowledge about stem cells and CSCs, focusing on mammary stem cells and breast CSCs, and their consequences for breast tumorigenesis and implications for breast cancer susceptibility, prognosis, and treatment.

Antibody-based identification of cell surface antigens: targets for cancer therapy

The identification of novel cell surface antigens present on tumor cells is crucial for developing new cancer therapies. Intact, viable cancer cells, which display cancer-restricted antigens in their native conformation and cellular context, provide a rich source of novel antigens. Antibody-based technologies are being used to probe the surface of intact cancer cells for cancer-specific antigen targets. In addition to identifying new proteins, these approaches are generating monoclonal antibodies (MAbs) to cancer-specific epitopes and nonprotein targets not amenable to genomics-based approaches. The multiple cell-based approaches developing epitope-specific MAbs to cancer antigens is likely to usher in a new era of therapeutic MAb target discovery.

Study of Cell Antigens and Intracellular DNA by Identification of Element-Containing Labels and Metallointercalators Using Inductively Coupled Plasma Mass Spectrometry

The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.

Andreas Rett and benign familial neonatal convulsions revisited

In 1964 Andreas Rett published the first account of a family with benign familial neonatal convulsions (BFNC). The authors retraced Rett's family and report that the clinical and genetic features of this original family fit the currently accepted definitions of BFNC. They also consider the career of Dr. Rett, a researcher and social reformer as well as an advocate for the rights of children with developmental disabilities.

Affinity ranking of antibodies using flow cytometry: Application in antibody phage display-based target discovery

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K D values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K D values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.

Primary cell cultures as models of prostate cancer development

This review focuses on primary cultures of human prostatic epithelial cells and their applications as models of normal and malignant biological behavior. Current abilities to culture cells from normal tissues, from premalignant dysplastic lesions (prostatic intraepithelial neoplasia), from primary adenocarcinomas, and from metastases are described. Evidence for representation of the interrelated cells of the normal prostatic epithelium--stem cells, basal epithelial cells, secretory epithelial cells, transit amplifying cells and neuroendocrine cells--in primary cultures is presented. Comparisons between normal and cancer-derived primary cultures are made regarding biological activities relevant to carcinogenesis, such as proliferation, apoptosis, differentiation, senescence, adhesion, migration, invasion, steroid hormone metabolism, other metabolic pathways and angiogenesis. Analyses of tumor suppressor activity, differential gene expression and cytogenetics in primary cultures have revealed changes relevant to prostate cancer progression. Preclinical studies with primary cultures have provided information useful for designing new strategies for chemoprevention, chemotherapy, cytotoxin therapy, radiation therapy, gene therapy and imaging. While the behavior of normal primary cultures is often used as a basis for comparison with established, immortal prostate cancer cell lines, the most informative studies are performed with donor-matched pairs of normal and malignant primary cultures, grown under identical conditions. Challenges that remain to be addressed if the full potential of primary cultures as a model system is to be realized include isolation, culture and characterization of stem cells, improved methodology to induce or maintain a fully differentiated, androgen-responsive phenotype, and identification of cell surface antigens or other markers with which to purify pure populations of live cancer or premalignant cells apart from non-malignant epithelial cells prior to culture.

Role of prostate stem cell antigen in prostate cancer research.

The identification of cell surface antigens is critical to the development of future prognostic and therapeutic modalities for the treatment of prostate cancer. Several prostate-specific proteins have been identified and are under investigation. This review reports on prostate stem cell antigen (PSCA), a protein with restricted expression that may have prognostic and therapeutic utility. PSCA is a glycosylphosphatidylinositol-anchored cell-surface protein belonging to the Ly-6/Thy-1 family of cell surface antigens, and a murine homologue has been described. It is expressed in the normal human prostate and overexpressed in human prostate cancers. Its overexpression has been correlated with increased Gleason score, advanced stage and bone metastasis. PSCA is co-amplified with MYC, an independent predictor of progression and death. PSCA may therefore be a useful predictor of tumor biology and a useful target of immunotherapy against prostate cancer. Evidence suggests a potential role in strategies employing cytotoxic T cell lymphocytes. Anti-tumor activity has been demonstrated with monoclonal antibodies in tumor take and established tumor xenograft models. Conjugated antibody has recently been reported to have anti-tumor activity in preclinical models. PSCA may serve as a tool in refining the prognosis of an individual cancer and may be a useful therapeutic target for immunotherapy. Future studies will be required to confirm its clinical utility as a prognostic factor. Future animal and clinical studies will be required to test various immunotherapy strategies for safety and efficacy. The study of PSCA regulation and expression may provide information on normal prostate development and prostate carcinogenesis.

Monoclonal antibody BB9 raised against bone marrow stromal cells identifies a cell-surface glycoprotein expressed by primitive human hemopoietic progenitors.

The identification of cell-surface antigens whose expression is limited to primitive hematopoietic progenitor cells (HPC) is of major value in the identification, isolation, and characterization of candidate stem cells in human hemopoietic tissues. Based on the observation that bone marrow stromal cells and primitive HPC share several cell-surface antigens, we sought to generate monoclonal antibodies to HPC by immunization with cultured human stromal cells. BALB/c mouse were immunized with human bone marrow (BM)-derived stromal cells. Splenocytes isolated from immunized mice were fused with the NS-1 murine myeloma cell line and resulting hybridomas selected in HAT medium, then screened for reactivity against stromal cells, peripheral blood (PB), and BM cells. A monoclonal antibody (MAb), BB9, was identified based on its binding to stromal cells, a minor subpopulation of mononuclear cells in adult human BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bound to a minor subpopulation of BM CD34(+) cells characterized by high-level CD34 antigen and Thy-1 expression, low-absent expression of CD38, low retention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cells, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stimulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstrated binding to CD34(+) cells from mobilized PB. Collectively, these data therefore demonstrate that MAb BB9 identifies an antigen, which is selectively expressed by hierarchically primitive human HPC and also by stromal cells.