The established method in prognosis of breast cancer includes detection of molecular markers, such as the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu. These markers are routinely checked via immunohistochemistry (IHC). HER-2/neu is also detected by fluorescent in situ hybridization (FISH). Flow cytometric analysis has not yet been used for detection of such markers. Flow cytometry was performed on four established breast cancer cell lines: MCF7, T47D, BT474, MDA-MB-231, and on one normal breast epithelial cell line: MCF10A. Flow cytometric analysis was used for the detection of ER, PR, HER-2/neu, epidermal growth factor receptor (EGFR), and E-cadherin. Currently, EGFR and E-cadherin are not standard predictive factors in determining survival of breast cancer patients, but both may be beneficial to prognosis. Cells undergoing flow cytometric analysis lost marker expression with increasing passage number. The highest expression was found at cells passaged 0-1 times. MCF7, T47D, and BT474 all had similar marker expression patterns. E-cadherin demonstrated a strongly positive pattern with marker expression of 85-92% among the three cell lines. ER, PR, and HER-2/neu demonstrated a weakly positive expression pattern when compared with E-cadherin. Marker expression ranged from 15 to 61%. These three cell lines were almost negative for expression of EGFR where expression ranged from 0 to 6%. MDA-MB-231 had almost no expression of all 5 markers, with positive values ranging from 0 to 5%. MCF10A had weak positive to almost negative expression values of ER, PR, HER-2/neu, and E-cadherin, which ranged from 3 to 13%. EGFR, both surface and cytoplasmic markers, again were not expressed in MCF10A cells with an expression value of <1%. We found that ER, PR, and HER-2/neu marker expressions in 5 out of 5 cell lines were consistent with established expression patterns. EGFR and E-cadherin expression in 4 out of 5 cell lines were also consistent with established expression patterns. We have shown that flow cytometry provides quantitative data on expression patterns of important prognostic markers in breast cancer.

10057 Methods of determining prognosis in breast, ovary, and colon cancer includes detection of molecular markers such as the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu. These markers are routinely assessed via immunohistochemistry (IHC) and Fluorescent In-situ hybridization (FISH). Flow cytometry (FC) has not yet been established for routine detection of these markers in tumors. We performed FC on cells from fourteen ovary, five breast, and four colon cancer tumors. FC was used to detect the presence of ER, PR, HER-2/neu, Epidermal Growth Factor Receptor (EGFR), E-cadherin, RAS, ERK and TUJ1. Cells obtained directly from tumoral tissue were simultaneous analysized by cell cycle stage (G0, G2M, and > G2M) and presence of antigenic markers. In ovarian tumors, 7 of 14 samples demonstrated >50% of cells with her-2/neu expression in aneuploid fractions (>G2M), 9 of 14 in G2M phase, and only 2 of 14 in G0 phase. With respect to ER, 7 of 14 ovarian tumors exhibited >19% of cells in G2M and >G2M fractions, while only 1 of 14 samples had >19% at G0. The PR receptor was expressed in >19% of cells in 8 of 14 (>G2M), 7 of 14 (G2M), and 2 of 14 (G0). In breast tumors, 4 of 5 tumors demonstrated >50% expression of HER-2/neu in G2M and >G2M cell phases, and only 2 of 5 demonstrated >50% her2neu expression in G0. Three of five tumors had >19% cellular expression of ER in the >G2M and G2M phases, while only one had >19% in G0. With PR, 3 of 5 breast tumors had >19% cellular expression in G0, G2M, and >G2M phases. In colon tumors, 2 of 4 had >50% cellular expression of her2neu in >G2M fractions, 4 of 4 in G2M, and only 1 of 4 in G0. Interestingly, cell fractions containing G2M and > G2M DNA had > 50% cellular expression for RAS and ERK in 2 of 4 colon tumors. No colon tumors expressed RAS and ERK significantly in G0 cells. We found that ER, PR, and HER-2/neu marker expression were consistent with established expression patterns. Additionally, these data point to specific expression patterns that are associated with the cell cycle stage. In particular, we note high expression levels of Her-2/nue, ER, and PR in aneuploid (>G2M) cell populations. These results suggest that patterns of differential expression can be readily assessed in tumor tissues using flow cytometry, and has the potential for broad application to the study of solid tumors. No significant financial relationships to disclose.

Idiopathic myelofibrosis (IM) is characterized by the constitutive mobilization of CD34(+) cells. IM peripheral blood (PB) CD34(+) cells had a reduced cloning efficiency and a lower frequency of cobblestone areas compared with normal granulocyte colony-stimulating factor (G-CSF)-mobilized PB CD34(+) cells. IM CD34(+) cells engrafted nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, demonstrating that they contain bone marrow (BM)-repopulating cells. G-CSF-mobilized CD34(+) cells produced multiple hematopoietic lineages within the NOD/SCID mice with a predominance of CD19(+) cells. By contrast, IM CD34(+) cells produced predominantly CD33(+) cells, increased numbers of CD41(+) cells, but fewer CD19(+) cells. Transcriptional clonality assays of the engrafted human IM cells demonstrated their clonal origin. CD34(+) cells from one patient isolated prior to leukemic transformation were capable of generating acute leukemia in NOD/SCID mice. The engrafted human cells exhibited the same abnormal karyotype as primary cells in a portion of the population. These findings demonstrate that BM-repopulating cells and more differentiated progenitor cells are constitutively mobilized into the PB in IM, and that their differentiation program is abnormal. In addition, the NOD/SCID model may be useful in gaining an understanding of the events occurring during the transition of IM to acute leukemia.

We are reporting the identification of a novel C-type lectin receptor–ligand pair that is involved in T cell costimulation. The receptor, OCILRP2/Clr-g, is rapidly induced following T cell activation and maintained at a substantial level of up to 72h. The ligand, NKRP1f, is predominantly expressed on dendritic cells (DC). The soluble OCILRP2-Ig blocking protein significantly suppresses specific antigen-stimulated T cell proliferation as well as IL-2 secretion both in vitro and in vivo; conversely, NKRP1f-expressing antigen presenting cells (APC) enhance B7.1/CD28-mediated costimulation for T cell proliferation through interaction with OCILRP2/Clr-g. Our studies reveal a unique functional interaction between two C-type lectins, OCILRP2/Clr-g and NKRP1f, during APC-mediated T cell costimulation and suggest a role for C-type lectins in maintaining T cell response or memory in vivo.

Given that expression of many genes changes when cells become malignant or are placed in. different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AM-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

The intracellular tyrosine kinase Brk is expressed in regenerating epithelial tissues with highest levels in the gastrointestinal tract and skin. In these tissues, Brk is restricted to epithelial cells that are exiting the cell cycle and undergoing terminal differentiation. While not expressed in mammary gland, Brk expression is often induced in primary breast tumors and breast tumor cell lines. To identify potential oncogenic functions of Brk, we utilized the immortalized Rat1a rat fibroblast cell line that is highly sensitive to transformation. We generated Rat1a cell lines that stably overexpress wild-type and activated Brk, and we analyzed their growth properties and ability to undergo apoptosis in response to stress and DNA damage. Overexpression of Brk did not induce anchorage-independent growth, and no changes in cell morphology or cell cycle progression were observed. However, when constitutively expressed, Brk sensitized Rat1a cells to a variety of apoptotic stimuli including serum deprivation and a combination of UV irradiation and serum starvation. These findings indicate that Brk does not promote proliferation of non-transformed cells, but plays a positive role in the regulation of the apoptotic response to DNA-damage and stress.

Crawling dendritic cells (CDCs) and herpes simplex virus-1 (HSV-1) amplicon vectors were utilized in this study: (1) to evaluate whether CDCs can be transduced by HSV-1 amplicon vectors; (2) to assess the effects of HSV-1 infections on structure and functions of CDCs; (3) to assess the capabilities of the transduced CDC to express, process, and present the transgene products; and (4) to induce in vitro and in vivo priming of T cells and B cells. CDC supported amplicon-mediated transgene expression while retaining the ability to perform mixed lymphocyte reaction (MLR) and priming of naive T cells. Then it was tested whether transduced CDC were able to initiate immunity ggainst either the amplicon particle and/or the product encoded by the delivered transgene by injecting groups of mice with transduced CDCs expressing GFP or LacZ. Spleen cells of these mice were stimulated by co-incubation with cells expressing: (1) either one of the transgenes (GFP or LacZ), (2) peptides of β-gal, or (3) peptides of HSV-1 glycoprotein B (gB). Interestingly, no significant cytotoxic T lymphocyte (CTL) activity against the transgenes or against gB was observed. In contrast, mice developed high levels of antibodies against gB and LacZ.

Although fluorescent dyes combined with flow cytometry have been used to confirm the viability of sperm in the past, methods to detect damage to spermatozoa following injury have been limited to use of dyes, which are often difficult to adequately compensate for in a single laser system.

Background: The characterization of human monocyte-derived dendritic cells (HM-DC) subsets have been a very difficult and elusive task because of the lack of appropriate reagents. We, therefore utilized several diverse approaches to evaluate two populations of HM-DC including flow cytometry, ultra-structural evaluation by electron microscopy, and functional assays. In addition, we studied the kinetics of the expression of antigens on HM-DC at diverse intervals of time and identify surface markers and functional differences of these two HM-DC subsets.Results: This study identified that a phenotype of HM-DC as defined by CD11c+, CD86+, and CD40+ could be separated in the presence or absence of TGF-beta1 into two different subsets of DC: (i) HM-DC without Birbeck granith (Mo-DC) and (ii) HM-DC with Birbeck granuli (Mo-LC). Furthermore, the functional studies showed that the HM-DC treated with TGF-beta1 (Mo-LC) exhibited the presence of Birbeck granuli and could actively divide. In addition, after undergoing more than four cell divisions, these cells split into at least two additional subsets of Mo-LC: (iia) Mo-LC with high forward scatter (FSC) and (iib) Mo-LC with normal FSC. In contrast, the Mo-DC cultured in absence of TGF-beta1 did not exhibit Birbeck granuli, showed reduced ability to divide, and kept the normal FSC when analyzed.Conclusions: This study enabled us to determine in HNI-DC: (i) the existence of antigenic and functional differences between various subpopulations of Mo-DC and Mo-LC; (ii) the existence of differences in the kinetics of antigens expression among the subsets of Mo-DC and Mo-LC; (iii) the existence of specific markers for each of the subpopulations of HM-DC. (C) 2004 Elsevier Inc. All rights reserved.

We have previously shown that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase inhibitor, trichostatin A (TSA) is capable of changing the fate of adult bone marrow CD34+ cells (Milhem M et al Blood 2004). We have now studied whether these drugs could alter the behavior of dividing CD34+CD90+ cord blood (CB) cells. The 5azaD/TSA treated cultures yielded 10 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines alone after 9 days of culture. The 5azaD/TSA treated cultures contained 2 fold greater numbers of colony forming cells (CFC) and 14 fold greater numbers of long-term (5wk) cobblestone area forming cells (CAFC) in comparison to culture containing cytokines alone. Although the CFC/CAFC plating efficiency of cells in cultures exposed to cytokines alone declined during the time of incubation, the cloning efficiency of cells exposed to 5azaD/TSA was equivalent to that of primary CD34+ cells. In order to determine the effects of cell division on the behavior of CD34+CD90+ cells in the 5azaD/TSA treated cultures, we utilized the cytoplasmic dye, CFSE. All of the CD34+CD90+ cells within the 5azaD/TSA pre-treated cultures divided at least once after 9 days of culture. 60% of the 5azaD/TSA treated CD34+CD90+ cells divided 5 times or more while 40% divided 1–4 times. The CD34+CD90+ cells lacking 5azaD/TSA pre-treatment underwent more cell divisions (90%, 5 or more divisions). The CD34+CD90+ cells pre-treated with 5azaD/TSA which had undergone 1-2 cell divisions had 11 fold greater numbers of CFU-Mix and 9 fold greater number of CAFC as compared to CD34+CD90+ cell population cultured in presence of cytokines alone. Furthermore CD34+CD90+ cells having 5 and more divisions had 4 fold more CFU-mix and 6.5 fold more CAFC in comparison to cells lacking 5azaD/TSA exposure. The CD34+CD90+ cells experiencing 1–4 divisions had 60 fold greater number of CFU-mix and 54 fold more CAFC in contrast to culture treated with cytokines alone. The in vivo SCID repopulating potential of CD34+CD90+ cells generated in presence or absence of 5azaD/TSA was then evaluated. When 5x104 CD34+CD90+ cells having undergone 1-2 cell divisions were re-isolated from 5azaD/TSA pre-treated cultures, all mice contained human hematopoietic cells. In addition, 1 of 3 mice transplanted with CD34+CD90+ cells (5x104) having undergone 3 and more cell divisions isolated from cultures pre-treated with 5azaD/TSA also displayed human hematopoietic engraftment. Furthermore 1 of 3 mice transplanted with equal numbers of the 5azaD/TSA pre-treated CD34+CD90+ cells having undergone 5 and more cell divisions also had evidence of human multilineage hematopoietic engraftment. By contrast, an equivalent number of CD34+CD90+ cells which had undergone more than 3 or more than 5 cell divisions from the cultures containing cytokines alone were incapable of engrafting NOD/SCID mice. These data suggest that the sequential addition of 5azaD and TSA ex vivo is not only capable of expanding the numbers of CD34+CD90+ cells and assayable progenitor cells but also capable of preserving their SCID repopulating potential. We conclude that 5azaD/TSA treatment of CD34+CD90+ cells results in their retention of the cellular program required to maintain their marrow repopulating potential despite their undergoing multiple cell divisions.

RATIONALE:This study aimed to expand the utilization of a simplified flow cytometric approach that employing SYBR-14/PI staining into broader flow cytometry applications, including (i) measurement of the DNA content; (ii) performing cell cycle analysis on mammalian cells; and (iii) sorting of live SYBR-14-stained mammalian cells based on DNA content.MATERIAL AND METHODS:Cell lines of human origin were stained with SYBR-14 and propidium iodide (PI) and assessed by a dual-color flow cytometry. Finally, sorting of living SYBR-14-stained human cell lines was performed.RESULTS:Dual staining with SYBR-14 and PI of human cells followed by flow cytometry analysis demonstrates that in addition to quality assessment, this staining could be utilized to determinate the DNA content on mammal cells. In addition, it resolves the diploid, tetraploid, and aneuploid DNA content. Furthermore, the SYBR-14-stained mammal cells were efficiently sorted based on DNA content and live cells were obtained. All these features have not been previously described with the utilization of this staining approach.CONCLUSIONS:Results of this study demonstrate that this flow cytometric approach not only allows assessment of the viability of cells, but also the DNA content of mammal cells. In addition, this approach allows one to sort viable cells stained with SYBR-14. These findings open-up unexpected and unrestricted avenues for sorting of living mammal cells and provide significant advantages over the traditionally cumbersome sorting approaches for living cells, which demand very specialized and expensive UV light sources as well as sophisticated sorting procedures.

ABSTRACT The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.

Dendritic cells initiate the immune response by presenting antigen in the context of varying levels of costimulation. The maturation state of the dendritic cell determines the quantity and quality (Th1, Th2) of the subsequent T cell response. Members of the NF-κB family of transcription factors have previously been implicated in dendritic cell development. Here, we used a mouse with a homozygous c-Rel deletion to investigate the role of c-Rel in the function of bone marrow derived dendritic cells. When direct presentation was evaluated, we found c-Rel−/− dendritic cells induce less allogeneic T cell stimulation than c-Rel+/+ dendritic cells. In addition, T cell encounters with c-Rel−/− dendritic cells generate less IFN-γ and IL-4 when compared to those with c-Rel+/+ DCs. A similar degree of functional compromise was observed in antigen-specific T cells that were stimulated by c-Rel−/− dendritic cells. Functional deficits were not linked to differences in the ability to undergo maturation per se, as LPS exposure induced similar morphologic and cell surface changes in both c-Rel+/+ and cRel−/− DCs. Although LPS induced a compensatory increase in the nuclear activity of fellow NF-κB family members, RelB and p65, LPS exposure was unable to negate the deficiencies in autologous T cell proliferation and cytokine production associated with the loss of c-Rel in dendritic cells. Taken together, our study supports a unique and non-redundant role for c-Rel in dendritic cell costimulatory capacity.

Interleukin-4 (IL-4) exhibits numerous biological and immunoregulatory functions on B- and T-lymphocytes, monocytes, and dendritic cells in both mice and humans. In the present study, we show that IL-4 also has a regulatory function in the cat species. Cells transfected with IL-4 DNA expressed a biologically active protein as demonstrated by the up-regulation of MHC class II molecules on B-lymphocytes (CD21(+)) in a flow cytometric assay. Increased levels of MHC class II expression on CD21(+) cells were seen in 11 out of 12 cats (p<0.05). In addition, 12 out of 12 cats showed up-regulation of MHC class II on CD21(-) cells, mainly consisting of T-lymphocytes (p<0.05). In contrast, concanavalin A (ConA)-induced culture supernatant from peripheral blood mononuclear cells (PBMCs) containing high levels of interferon-gamma (IFN-gamma) transcripts induced down-regulation of MHC class II molecules on CD21(+) cells of all samples (p<0.05). Variable results were observed for CD21(-) cells incubated with ConA-conditioned medium (p=0.71). The nature of the cytokine(s) responsible for these effects remains to be determined. However, the fact that down-regulation of MHC class II molecules on B cells occurred in all cats tested suggests that IFN-gamma may be involved. These data provide further insight into the mechanism by which MHC class II expression is regulated in feline lymphocytes, and suggest that the Th1/Th2 paradigm is also present in the cat.

Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host’s immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ–eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.

BACKGROUND In this study, we compared herpes simplex virus type 1 (HSV-1) amplicon vector stocks prepared by transient cotransfection with two different BAC-cloned packaging-defective HSV-1 helper genomes, fHSVDeltapacDelta27 and fHSVDeltapac, with respect to transduction efficiency and cytotoxicity. Both fHSVDeltapacDelta27 and fHSVDeltapac are packaging defective because the pac signals have been deleted; fHSVDeltapacDelta27 contains an additional deletion in the HSV-1 ICP27 gene, which increases the safety of the system. METHODS HSV-1 amplicon pHSVGFP under the control of the HSV-1 immediate-early (IE) 4/5 promotor was packaged into virus particles by transient cotransfection with either fHSVDeltapacDelta27 or fHSVDeltapac DNA. Cultures were infected with the two different vector stocks and examined under the fluorescence microscope and analyzed by flow cytometry over a 5-day period to assess transduction efficiency and cytotoxicity. RESULTS Both vector stocks, pHSVGFP[fHSVDeltapacDelta27] and pHSVGFP[fHSVDeltapac], efficiently transduced the target cells. Interestingly, the highest mean fluorescence intensities were measured at 1 day after infection, whereas the number of GFP-fluorescent cells reached a peak at day 3 after infection. At day 3 after infection, a slight increase in the number of dead cells was observed in those cultures transduced with high doses of vector stock. Between days 3 and 4 after infection, the number of dead cells increased dramatically in all the cultures, transduced and nontransduced. Only the cultures infected with a high dose of pHSVGFP[fHSVDeltapac] displayed a significant further increase in the number of dead cells between days 4 and 5 postinfection. CONCLUSIONS Flow cytometry allowed comparison of transduction efficiency and cytotoxicity mediated by the two different amplicon vector stocks. Cultures infected with pHSVGFP[fHSVDeltapacDelta27] were more viable than those infected with pHSVGFP[fHSVDeltapac](P < 0.05). The practical implications of this study are at the level of vector design. Flow cytometry has proven a fast and reliable approach to assess the quality of potential gene transfer vectors prior to their use in (pre) clinical trials.