Laughing it off? Humour, affect and emotion work in communities living with nuclear risk.

Over the past two decades, an increasing number of risk researchers have recognized that risks are not simply objective hazards but that the meanings of risk are discursively negotiated, dynamic and embedded within the wider social relations that constitute everyday life. A growing interest in the complexity and nuances of risk subjectivities has alerted sociocultural researchers not only to what is said in a risk situation, but also to how it is said and to what is unsaid and even, in a particular context, unsayable; to the intangible qualities of discourse that communicate additional meanings. Humour is both an intangible and marks such intangible meanings, yet it has largely been ignored and insufficiently theorized by risk researchers. In this paper, we draw upon insights from the humour literature - suspending the belief that humour is inherently good - to analyse and theorize humour as a way of examining the meanings and functions of risk. We show how humour can both mask and carefully reveal affectively charged states about living with nuclear risk. As such, it helps risk subjects to live with risk by suppressing vulnerabilities, enabling the negotiation of what constitutes a threat, and engendering a sense of empowerment. We conclude that humorous talk can be serious talk which can enrich our understandings of the lived experience of risk and of risk subjectivities.

Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.

Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation.

OBJECTIVE: The effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on their osteogenic differentiation. METHODS: Murine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)(+) CD31(-) CD45(-) population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1beta and TNFalpha. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR). RESULTS: We show that osteogenic differentiation from the MSC population is suppressed by IL-1beta and TNFalpha. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, alpha1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFalpha inhibited osteonectin and osteopontin mRNA expression and only IL-1beta reduced cell proliferation. CONCLUSIONS: The convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1beta and TNFalpha, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC.

Reframing nuclear power in the UK energy debate: nuclear power, climate change mitigation and radioactive waste.

In the past decade, human influence on the climate through increased use of fossil fuels has become widely acknowledged as one of the most pressing issues for the global community. For the United Kingdom, we suggest that these concerns have increasingly become manifest in a new strand of political debate around energy policy, which reframes nuclear power as part of the solution to the need for low-carbon energy options. A mixed-methods analysis of citizen views of climate change and radioactive waste is presented, integrating focus group data and a nationally representative survey. The data allow us to explore how UK citizens might now and in the future interpret and make sense of this new framing of nuclear power--which ultimately centers on a risk-risk trade-off scenario. We use the term "reluctant acceptance" to describe how, in complex ways, many focus group participants discursively re-negotiated their position on nuclear energy when it was positioned alongside climate change. In the concluding section of the paper, we reflect on the societal implications of the emerging discourse of new nuclear build as a means of delivering climate change mitigation and set an agenda for future research regarding the (re)framing of the nuclear energy debate in the UK and beyond.

Increased proteolysis. An effect of increases in plasma cortisol within the physiologic range.

Prolonged exposure to glucocorticoids in pharmacologic amounts results in muscle wasting, but whether changes in plasma cortisol within the physiologic range affect amino acid and protein metabolism in man has not been determined. To determine whether a physiologic increase in plasma cortisol increases proteolysis and the de novo synthesis of alanine, seven normal subjects were studied on two occasions during an 8-h infusion of either hydrocortisone sodium succinate (2 micrograms/kg X min) or saline. The rate of appearance (Ra) of leucine and alanine were estimated using [2H3]leucine and [2H3]alanine. In addition, the Ra of leucine nitrogen and the rate of transfer of leucine nitrogen to alanine were estimated using [15N]leucine. Plasma cortisol increased (10 +/- 1 to 42 +/- 4 micrograms/dl) during cortisol infusion and decreased (14 +/- 2 to 10 +/- 2 micrograms/dl) during saline infusion. No change was observed in plasma insulin, C-peptide, or glucagon during either saline or cortisol infusion. Plasma leucine concentration increased more (P less than 0.05) during cortisol infusion (120 +/- 1 to 203 +/- 21 microM) than saline (118 +/- 8 to 154 +/- 4 microM) as a result of a greater (P less than 0.01) increase in its Ra during cortisol infusion (1.47 +/- 0.08 to 1.81 +/- 0.08 mumol/kg X min for cortisol vs. 1.50 +/- 0.08 to 1.57 +/- 0.09 mumol/kg X min). Leucine nitrogen Ra increased (P less than 0.01) from 2.35 +/- 0.12 to 3.46 +/- 0.24 mumol/kg X min, but less so (P less than 0.05) during saline infusion (2.43 +/- 0.17 to 2.84 +/- 0.15 mumol/kg X min, P less than 0.01). Alanine Ra increased (P less than 0.05) during cortisol infusion but remained constant during saline infusion. During cortisol, but not during saline infusion, the rate and percentage of leucine nitrogen going to alanine increased (P less than 0.05). Thus, an increase in plasma cortisol within the physiologic range increases proteolysis and the de novo synthesis of alanine, a potential gluconeogenic substrate. Therefore, physiologic changes in plasma cortisol play a role in the regulation of whole body protein and amino acid metabolism in man.

Perturbed hemopoiesis and the generation of multipotential stem cell clones in src-infected bone marrow cultures is an indirect or transient effect of the oncogene.

Multipotential stem cell lines, derived specifically from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures.

Aspirin reduces cutaneous flushing after administration of an optimized extended-release niacin formulation.

OBJECTIVE: Niacin is an effective treatment for dyslipidemia due to its favorable effects on multiple lipid parameters. Clinical utility of niacin is sometimes limited, however, because of cutaneous flushing. A once-daily, extended-release (ER) niacin formulation has been shown to significantly reduce flushing compared to immediate-release niacin. An optimized (reformulated) version of niacin ER has recently been developed and was shown in a previous study to significantly reduce flushing intensity (severity) compared to the non-optimized (commercial) formulation. The current study was designed to evaluate the effect of aspirin on various indices of flushing when administered with the optimized niacin ER formulation. METHOD: This was a randomized, double-blind, double-dummy, placebo-controlled flush provocation crossover study in healthy males. To increase the probability of flushing, subjects received a single dose of reformulated niacin ER 2,000 mg, which is the upper limit of the approved dosage range. Subjects received 650 mg aspirin orally either 30 minutes before or concomitantly with niacin ER, or placebo with niacin ER, in 3-way crossover fashion. The primary endpoint was the number of subjects who reported at least one flushing event. Secondary endpoints included the perceived intensity and duration of flushing symptoms. RESULTS: In the 148 men who completed all treatments, aspirin significantly reduced flushing incidence (the primary endpoint) following administration of niacin ER compared with placebo. Among subjects receiving placebo, 77% of subjects reported flushing with niacin ER. Among subjects receiving aspirin, 53-61% of subjects reported flushing (pretreatment and concomitant treatment, respectively, both p < 0.001 compared with placebo) with niacin ER. Aspirin also significantly reduced intensity and duration of flushing (by 30-40%) compared with no aspirin. The two aspirin-containing treatments (i.e. pre- or concomitant treatment) were similar in their effects on flushing incidence, intensity and duration. Median intensity on a 100 mm visual analogue scale (VAS) was reduced from 33 mm with placebo to 19-23 mm with aspirin. Median duration was reduced from approximately 1 hour with placebo to 37-48 minutes with aspirin. CONCLUSION: Aspirin significantly reduced the incidence, intensity and duration of flushing associated with reformulated niacin ER. These results support the administration of aspirin prophylactically to decrease niacin-induced cutaneous flushing and to improve patient adherence and acceptability of chronic niacin treatment at therapeutic doses.

Seeing what is coming: building collision-sensitive neurones.

The image of a rapidly approaching object has to elicit a quick response. An animal needs to know that the object is approaching on a collision course and how imminent a collision is. The relevant information can be computed from the way that the image of the object grows on the retina of one eye. Firm data about the types of neurones that react to such looming stimuli and trigger avoidance reactions come from recent studies on the pigeon and the locust. The neurones responsible are tightly tuned to detect objects that are approaching on a direct collision course. In the pigeon these neurones signal the time remaining before collision whereas in the locust they have a crucial role in the simple strategy this animal uses to detect an object approaching on a collision course.

Improved control of niacin-induced flushing using an optimized once-daily, extended-release niacin formulation.

INTRODUCTION: Niacin is a recognized treatment for dyslipidemia due to its favorable effects on all lipid parameters. However, the clinical use of niacin has been limited by its adverse effects, particularly cutaneous flushing. A newly reformulated 1,000 mg niacin ER tablet has been designed to reduce flushing relative to the original commercial niacin ER formulation. The aim of this study is to compare the incidence, intensity and duration of flushing between the 1,000 mg reformulated niacin ER and the 1,000 mg commercially available formulation, when administered as a single 2,000 mg dose to healthy male volunteers. METHODS: This was a double-blind, double-dummy, placebo-controlled, 3-way crossover, flush provocation study conducted at a single center. To increase the probability of flushing, subjects were administered niacin ER at the upper limit of the approved dosage range (2,000 mg), and were precluded from using aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs) during the study. Subjects received reformulated niacin ER, commercial niacin ER or placebo in a 3-way crossover fashion. The primary flushing variable was the occurrence of a flushing event. Secondary flushing variables included the number of flushing episodes, intensity and duration of flushing for both overall flushing events and for individual symptoms of flushing (redness, warmth, tingling and itching). RESULTS: A total of 156 subjects were enrolled in the study. Of 133 subjects who received at least 1 dose of study medication in at least 2 study periods, 89% of subjects experienced flushing during treatment with reformulated niacin ER, and 98% of subjects experienced flushing during treatment with commercial niacin ER. This difference was statistically significant (p - 0.0027). Reformulated niacin ER resulted in a 42% reduction in median flush intensity (p < 0.0001) and a 43% reduction in median flush duration (p < 0.0001) relative to commercial niacin ER. The duration of first flushing event was more than 1 hour shorter with reformulated niacin ER. During the study, 29% of subjects (45/156) experienced treatment-emergent adverse events, which were mostly mild in intensity and considered to be remotely related or unrelated to the study drug. CONCLUSION: The 1,000 mg reformulated niacin ER tablet substantially decreases the incidence, intensity and duration of flushing relative to the commercially available 1,000 mg niacin ER tablet, and represents an improved niacin therapy option.

Adolescents' knowledge of and attitudes toward Minnesota laws concerning adolescent medical care.

BACKGROUND: Healthcare delivery to adolescents involves consent to care, confidentiality, and reimbursement in attendance to patients' medical problems. State laws address the medical care of minors with respect to privacy and autonomy. This study was conducted to determine adolescents' knowledge and attitudes toward the laws in their state, Minnesota, that influence their medical care. This information may guide health care providers in their education of adolescent patients, as well as advise future legislation on the healthcare of minors. METHODS: Written questionnaires were administered to 636 9(th) through 12(th) grade students in required classes at the three public high schools in Rochester, MN, providing a representative sample of approximately fifty participants from each of the four grades. Results from the sixteen-question, anonymous survey were compiled to calculate an overall median knowledge score. Median scores were also reported by individual question, policy category, gender, grade, and socioeconomic status. Adolescent opinion was scored on a system in which +1 signified a "good law," 0 "neither a good nor bad law," and -1 a "bad law." These opinion scores were used to determine adolescents' attitudes by category as well as an overall positive, negative, or neutral opinion toward the laws. RESULTS: 594(93.4%) students returned surveys. Adolescents obtained a median score of 31.3% (range, 0-100%) correct on a knowledge test. Opinion score was positive, with a median of + 0.38. 81.1% of adolescents reported that the laws in the survey had no effect on their lives. CONCLUSIONS: These results indicate that adolescents are not knowledgeable of Minnesota laws regarding their medical care. Overall opinion of the existing laws is positive, particularly of those laws that protect minor consent. Most adolescents feel that these laws have not affected their lives. Low knowledge and a lack of a sense of impact from these laws indicate a need for clinicians to educate patients and their families about the legal aspects of adolescent medical care. These results are particularly relevant at a time when changes to current minor health care legislation are under consideration.

MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies.

Despite the importance of bone marrow stromal cells in hemopoiesis, the profile of surface molecule expression is relatively poorly understood. Mice were immunized with cultured human bone marrow stromal cells in order to raise monoclonal antibodies to novel cell surface molecules, which might be involved in interactions with hemopoietic cells. Three antibodies, WM85, CC9 and EB4 were produced, and were found to identify a 100-110 kDa antigen on bone marrow fibroblasts. Molecular cloning revealed the molecule to be MUC18 (CD146), a member of the immunoglobulin superfamily, previously described as a marker of metastatic melanoma. In addition to the expected expression on melanoma cell lines and endothelial cells, a number of human leukemic cell lines were found to express MUC18, including all six T leukemia lines tested, one of five B lineage lines and one of four myeloid lines. Analysis of bone marrow samples from patients revealed positivity in 20% of B lineage ALL (n = 20), one of three T-ALL, 15% of AML (n = 13) and 43% of various B lymphoproliferative disorders (n = 7). No apparent reactivity was observed with mononuclear cells from normal peripheral blood or bone marrow, including candidate hemopoietic stem cells characterized by their expression of the CD34 antigen. However, positive selection of bone marrow mononuclear cells labeled with MUC18 antibody revealed a rare subpopulation (<1%) containing more than 90% of the stromal precursors identified in fibroblast colony-forming assays. The structure and tissue distribution of MUC18 suggest a functional role in regulation of hemopoiesis.

Ethical considerations in the early composite tissue allograft experience: a review of the Louisville Ethics Program.

This paper reviews the formulation and evolution of the ethical component in one of the earliest clinical composite tissue allograft (CTA) programs, the hand transplantation program in Louisville, Kentucky, USA. The purpose was to derive lessons and define principles to give guidance for future programs and introduction of new CTA. We reviewed the initial ethical considerations, including input from respected ethical scholars, guidelines for innovative procedures transparency in public and professional scrutiny, and compliance with human studies regulations (IRB approval). We found the initial focus on ethics, scholarly input, guidelines for innovative procedures, and human studies protection regulations to be valid. Moreover, we noted the effect of autonomy in subjective, quality-of-life benefits on equipoise and effective risk-benefit analysis in effective informed consent. We found that psychiatric screening and support to be exceptionally valuable in protecting autonomy, suitability for participation, assessing personality organization, and determining compliance ability. We conclude that the program ethical principles were validated. For future CTA programs and procedures, we recommend an ethical emphasis with adherence to high standards and transpire to independence to scrutiny and oversight. We recommend protection of autonomy judgments in equipoise judgment and informed consent. We recommend skilled psychiatric screening and support. We endorse scholarship, scientific accuracy, and data sharing.

Cytoskeleton and integrin-mediated adhesion signaling in human CD34+ hemopoietic progenitor cells.

Significant progress has been made recently in the understanding of cell adhesion signaling. Many components of focal adhesion complexes have been identified in fibroblasts and endothelial cells, showing considerable overlap and complementarity between growth signaling mediated by growth factor receptors and adhesive signaling mediated by cell adhesion receptors such as integrins. These studies showed that the cytoskeleton is essential for the correct intracellular localization of large signaling complexes that regulate the cellular machinery. Although adhesive interactions are essential to maintain steady-state hemopoiesis, the study of the function and role of adhesive interactions in hemopoietic progenitor and stem cells is less advanced. As in fibroblasts, functional overlap between hemopoietic growth factor receptors and cell adhesion receptors has been demonstrated, with the cytoskeleton likely playing a critical role in integrating information provided by soluble factors and cell adhesion molecules constituting the hemopoietic microenvironment. The intention of this article is to give a critical review of the current knowledge about the cytoskeleton and integrin-mediated signaling in hemopoietic progenitor cells.

Expression of the Wilms' tumor gene (WT1) in normal hemopoiesis.

The Wilms tumor suppressor gene (WT1) is mutated in a number of cases of Wilms' tumor as well as in mesothelioma and leukemia. It encodes a transcription factor derived from any one of four alternate transcripts. WT1 has a restricted pattern of expression within the body and within the hemopoietic system its expression is limited to primitive leukemias and a number of leukemic cell lines. Given the overexpression of WT1 in leukemias, we have addressed the question of whether this gene is expressed within the normal hemopoietic system. Mononuclear bone marrow (BM) cells obtained from normal donors were separated by fluorescence-activated cell sorting (FACS) into "primitive" (CD34+) and "mature" (CD34-) cell populations. Total RNA extracted from these cells was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using primers based on the WT1 sequence, to examine the expression of this gene within the hemopoietic system. Phenotypic purity of cells was guaranteed by performing single-cell sorting followed by RT-PCR to define the precise cellular phenotypes that express WT1. Expression of WT1 was detected in cells bearing the CD34+ phenotype but not in those cells lacking expression of CD34. In addition, single-cell analysis revealed that expression of WT1 occurred in the candidate stem cell-containing population of hemopoietic cells which have the phenotype CD34+ CD38-. Moreover, the single-cell RT-PCR analysis also demonstrated that differential expression of alternate transcripts of WT1 occurs between hemopoietic progenitor cells with the same phenotype. In conclusion, expression of WT1 is limited to early progenitors of the blood system, which suggests that this gene plays a critical role in hemopoietic development.

c-kit is expressed by primitive human hematopoietic cells that give rise to colony-forming cells in stroma-dependent or cytokine-supplemented culture.

Using monoclonal antibody (MAB) YB5.B8, we have examined the expression of the c-kit protein, the receptor for the hematopoietic cytokine stem cell factor (SCF), on primitive hematopoietic cells. Bone marrow mononuclear cells (BMMNC) enriched for immature cells by differential agglutination using the lectin soybean agglutinin (SBA) were subjected to multiparameter fluorescence activated cell sorting (FACS) based on light-scattering properties, the expression of the c-kit protein and the CD34 antigen, and the retention of the vital fluorescent dye, Rhodamine 123 (Rh123). Sorted populations were assayed for their content of directly clonogenic progenitor cells (colony-forming units-granulocyte/macrophage [CFU-GM], burst-forming units-erythroid [BFU-E], and multipotential colony-forming units [CFU-Mix]) and for the presence of more primitive progenitor cells ("pre-CFU"). The latter were assayed by (1) their ability to initiate and sustain hematopoiesis in a standard stromal cell-dependent culture system and (2) their capacity for de novo generation of clonogenic progenitors in response to a combination of six recombinant hematopoietic cytokines in a stroma-independent suspension culture assay. A mean of 76% of CD34+ cells were found to coexpress c-kit. The majority of directly clonogenic cells (98% of CFU-GM, 98% of CFU-Mix, and 85% of BFU-E) were found in the CD34+c-kit+ fraction. Similarly, all pre-CFU were recovered in the CD34+c-kit+Rh123dull fraction, irrespective of whether the cells were maintained on marrow stromal cells or in cytokine-supplemented liquid culture. A mean of 87% (range 70-100%) of the CD34+Rh123dull cells also expressed c-kit. Since SCF has been reported to act as a growth factor for early lymphoid cells as well as myeloid cells, we looked for coexpression of c-kit and early lymphoid markers in the CD34+ population by multiparameter flow cytometry. Coexpression of c-kit on a minority of cells with markers of B or T lineages was observed. The majority of early lymphoid cells, however, appeared to lack c-kit expression. This was confirmed by the finding that only 4% of c-kit+CD34+ cells showed terminal deoxynucleotidyl transferase (TdT) activity, compared with 25% of the c-kit-CD34+ cells.

High-level expression of a novel epitope of CD59 identifies a subset of CD34+ bone marrow cells highly enriched for pluripotent stem cells.

To further define the hierarchy of human hematopoietic progenitor cells, we have attempted to identify antibodies to cell-surface molecules expressed on CD34+ progenitor cell subsets. Herein we describe the utility of a new monoclonal antibody, HCC-1, which binds to a novel epitope of CD59 differentially expressed among CD34+ progenitor cells. HCC-1 subdivides the adult marrow CD34+ population into HCC-1high and HCC-1low/- fractions of approximately equal size. Cobblestone area-forming cells (CAFC) in long-term bone marrow culture were enriched 10-30-fold in CD34+HCC-1high cells compared with CD34+HCC1-low/- cells and two-fold compared with CD34+ cells. When injected into fetal human bone fragments implanted in SCID mice, the CD34+HCC-1high population showed potent engrafting activity leading to the production of myeloid, lymphoid, and erythroid elements, as well as the retention of progenitor cell phenotype. These studies demonstrate that the CD34+HCC-1high population contains primitive pluripotent hematopoietic stem cells. No hematopoietic engrafting activity was detected in the CD34+HCC-1low/- population. Consistent with this finding, simultaneous five-color flow cytometric analysis revealed that HCC-1high cells include virtually all CD34+Thy-1+Lin- cells, a cell population previously characterized as highly enriched for primitive pluripotent hematopoietic stem cells. The ability of CD34+ cells divided into subsets by HCC-1 to produce T cells was assessed by transplantation of sorted cells into human fetal thymus implanted into SCID mice. A higher frequency of thymus-engrafting activity was observed in the CD34+HCC-1high than in the CD34+HCC-1low/- population. Consistent with the limited ability to engraft in the SCID-hu thymus model, the CD34+HCC-1low/- population was shown to contain a low frequency of CD34+CD10+ lymphoid progenitor cells. We conclude that the HCC-1 epitope is expressed at high levels on a subset of CD34+ cells that contain virtually all primitive pluripotent hematopoietic stem cells and that the population of CD59 molecules expressed on CD34+ cells is not homogeneous.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A novel epitope of CD59 expressed by primitive human hematopoietic progenitors.

OBJECTIVE: The aim of this study was to determine the identity of the cell surface molecule on primitive hematopoietic cells recognized by monoclonal antibody HCC-1. MATERIALS AND METHODS: Screening of a cDNA expression library prepared from human bone marrow stromal cells with HCC-1 yielded a single cDNA, which when expressed in FDCP-1 cells, resulted in the specific acquisition of HCC-1 binding. The cDNA demonstrated complete identity with CD59, a phosphoinositol glycan-linked membrane protein that protects cells against autologous complement attack. The ubiquitous expression of CD59 is in marked contrast to the restricted reactivity of HCC-1. Studies were performed to examine the basis for the novel specificity of HCC-1 for CD59. The epitope on CD59 identified by HCC-1 was mapped using a series of rat/human CD59 chimeric proteins. Immunoprecipitation analyses were performed to determine whether CD59 associates with other membrane proteins. RESULTS: Mutagenesis of Asn18 did not alter the binding of HCC-1 to CD59, suggesting that N-linked carbohydrates are not responsible for the binding specificity of HCC-1. The epitope for HCC-1 was shown to differ from that identified by previously described CD59 antibodies, encompassing residues A31, L33, R55, and L59. An 80 kDa protein co-immunoprecipitated with CD59 in the HCC-1(-) cell line HL-60 but not in HCC-1(+) K562 cells. CONCLUSION: Collectively, these data support the hypothesis that the unique specificity of HCC-1 for CD59 is due in part to recognition of a novel epitope, which is masked as a result of association with an as yet unidentified 80 kDa protein.

Integrin expression and function on human osteoblast-like cells.

The integrin family of cell adhesion molecules are a series of cell surface glycoproteins that recognize a range of cell surface and extracellular matrix (ECM)-associated ligands. To date, the precise role of individual integrin molecules in bone cell-ECM interactions remains unclear. Cell binding assays were performed to examine the ability of normal human bone cells (NHBCs) to adhere to different ECM proteins in vitro. NHBCs displayed preferential adhesion to fibronectin over collagen types I, IV, and vitronectin and showed low affinity binding to laminin and collagen type V. No binding was observed to collagen type III. The integrin heterodimers alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and alpha v beta 5 were found to be constitutively expressed on the cell surface of NHBCs by flow cytometric analysis. The integrins alpha 4 beta 1 and alpha 6 beta 1 were not expressed by NHBCs. Subsequent binding studies showed that NHBC adhesion to collagen and laminin was mediated by multiple integrins where cell attachment was almost completely inhibited in the presence of a combination of function-blocking monoclonal antibodies (Mabs) to alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, and beta 1. In contrast, the adhesion of NHBCs to fibronectin was only partially inhibited (50%) in the presence of blocking Mabs to alpha 3 beta 1, alpha 5 beta 1, and beta 1. The attachment of NHBCs to collagen, laminin, fibronectin, and vitronectin was also found to be unaffected in the presence of a function-blocking Mab to alpha v beta 3. The results of this study indicate that beta 1 integrins appear to be the predominant adhesion receptor subfamily utilized by human osteoblast-like cells to adhere to collagen and laminin and in part to fibronectin.