Probing Catalyst Function - Electronic Modulation of Chiral Polyborate Anionic Catalysts.

Boroxinate complexes of VAPOL and VANOL are a chiral anionic platform that can serve as a versatile staging arena for asymmetric catalysis. The structural underpinning of the platform is a chiral polyborate core that covalently links together alcohols (or phenols) and vaulted biaryl ligands. The polyborate platform is assembled in situ by the substrate of the reaction, and thus a multiplex of chiral catalysts can be rapidly assembled from various alcohols (or phenols) and bis-phenol ligands for screening of catalyst activity. In the present study, variations in the steric and electronic properties of the phenol/alcohol component of the boroxinate catalyst are probed to reveal their effects on the asymmetric induction in the catalytic asymmetric aziridination reaction. A Hammett study is consistent with a mechanism in which the two substrates are hydrogen-bonded to the boroxinate core in the enantiogenic step. The results of the Hammett study are supported by a computational study in which it is found that the H-O distance of the protonated imine hydrogen bonded to the anionic boroxinate core decreases with an increase in the electron releasing ability of the phenol unit incorporated into the boroxinate. The results are not consistent with a mechanism in which the boroxinate catalyst functions as a Lewis acid and activates the imine by a Lewis acid/Lewis base interaction.

Treatment of supragastric belching with cognitive behavioral therapy improves quality of life and reduces acid gastroesophageal reflux.

OBJECTIVES: Excessive supragastric belching (SGB) manifests as troublesome belching, and can be associated with reflux and significant impact on quality of life (QOL). In some GERD patients, SGB-associated reflux contributes to up to 1/3 of the total esophageal acid exposure. We hypothesized that a cognitive-behavioral intervention (CBT) might reduce SGB, improve QOL, and reduce acid gastroesophageal reflux (GOR). We aimed to assess the effectiveness of CBT in patients with pathological SGB. METHODS: Patients with SGB were recruited at the Royal London Hospital. Patients attended CBT sessions focused on recognition of warning signals and preventative exercises. Objective outcomes were the number of SGBs, esophageal acid exposure time (AET), and proportion of AET related to SGBs. Subjective evaluation was by patient-reported questionnaires. RESULTS: Of 51 patients who started treatment, 39 completed the protocol, of whom 31 had a follow-up MII-pH study. The mean number of SGBs decreased significantly after CBT (before: 116 (47-323) vs. after 45 (22-139), P<0.0003). Sixteen of 31 patients were shown to have a reduction in SGB by >50%. In patients with increased AET at baseline, AET after CBT was decreased: 9.0-6.1% (P=0.005). Mean visual analog scale severity scores decreased after CBT (before: 260 (210-320) mm vs. after: 140 (80-210) mm, P<0.0001). CONCLUSIONS: Cognitive behavioral therapy reduced the number of SGB and improved social and daily activities. Careful analysis of MII-pH allows identification of a subgroup of GERD patients with acid reflux predominantly driven by SGB. In these patients, CBT can reduce esophageal acid exposure.

Pyro-Borates, Spiro-Borates, and Boroxinates of BINOL-Assembly, Structures, and Reactivity.

VANOL and VAPOL ligands are known to react with three equivalents of B(OPh)3 to form a catalytic species that contains a boroxinate core with three boron atoms, and these have proven to be effective catalysts for a number of reactions. However, it was not known whether the closely related BINOL ligand will likewise form a boroxinate species. It had simply been observed that mixtures of BINOL and B(OPh)3 were very poor catalysts compared to the same mixtures with VANOL or VAPOL. Borate esters of BINOL have been investigated as chiral catalysts, and these include meso-borates, spiro-borates, and diborabicyclo-borate esters. Borate esters are often in equilibrium, and their structures can be determined by stoichiometry and/or thermodynamics, especially in the presence of a base. The present study examines the structures of borate esters of BINOL that are produced with different stoichiometric combinations of BINOL with B(OPh)3 in the presence and absence of a base. Depending on conditions, pyro-borates, spiro-borates, and boroxinate species can be generated and their effectiveness in a catalytic asymmetric aziridination was evaluated. The finding is that BINOL borate species are not necessarily inferior catalysts to those of VANOL and VAPOL but that, under the conditions, BINOL forms two different catalytic species (a boroxinate and a spiro-borate) that give opposite asymmetric inductions. However, many BINOL derivatives with substitutents in the 3- and 3'-positions gave only the boroxinate species and the 3,3'-Ph2BINOL ligand gave a boroxinate catalyst that gives excellent inductions in the aziridination reaction. BINOL derivatives with larger groups in the 3,3'-position will not form either spiro-borates or boroxinate species and thus are not effective catalysts at all.

Mesenchymal stem cells from CD34(-) human umbilical cord blood.

Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34(-) UCB cells. CD34(+) cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34(-) cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic-fibroblast growth factor. Isolated CD34(+) cells were also cultured in the same medium. Adherent fibroblast-like cells at passage 3-4 were analyzed by fluorescence-activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4-5 weeks in culture, the cells from the CD34(-) fraction became confluent with flat and fibroblast-like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation-activated receptor gamma-2 (PPARgamma2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox-9 genes. In contrast, cells from the CD34(+) fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34(-) fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

La investigación en Enfermería: Una mirada desde el enfoque de Género / Nursing research: a view from the gender focus

A partir de algunos ejemplos, se evidencia la limitada participación de las mujeres en la actividad científica y se fundamenta cómo esta exclusión tiene su base en decisiones sociales androcéntricas y no en "limitaciones" biológicas. Se destaca la importancia del enfoque de género para hacer visible la discriminación a las mujeres en diversos ámbitos del hacer humano, entre ellos la ciencia. Finalmente, se traspolan estos argumentos al caso concreto de la investigación en la Enfermería y se hacen algunas propuestas viables de ser llevadas a cabo, entre otras, en la Escuela Nacional de Enfermería y Obstetricia de la UNAM.

Considering the corresponding history, androcentric decisions, not biological "limitations", have been the base for the exclusion of women from social activities such as science. Here, the perspective of gender is highlighted as one bridge to make this discrimination clearly visible. Also, the specific case of Nursing Research is considered by the National School of Nursing and Obstetrics (ENEO) of the National Autonomous University of Mexico (UNAM) along with some proposals for possible solutions.

La violencia familiar en San Luis Tlaxialtemalco: un estudio con enfoque de género / Family violence in San Luis Tlaxialtemalco: a gender focused study

Este trabajo de investigación se centró en el objetivo: de identificar y describir la violencia familiar en un grupo de mujeres vecinas y usuarios del Centro Comunitario de Atención Primaria a la Salud (CCOAPS) en San Luis Tlaxialtemalco (SLT) México, de la ENEO-UNAM. Metodología: Mediante una investigación descriptiva y transversal, de corte cualitativo, con perspectiva de género, llevada a cabo en el espacio físico del CCOAPS de mayo del 2000 a mayo del 2001, de manera convencional se entrevistó a profundidad a 15 mujeres adultas entre 18 y 60 años de edad que acudieron en demanda de atención, que vivían o habían vivido en pareja y que aceptaron ser entrevistadas, en forma privada y anónima. Se identificaron las categorías presentes en la construcción social de modelos estereotipados de poder y dominio para unos y de tolerancia para otras, a fin de elaborar una guía de entrevista integrada con 11 argumentos sobre "lo esperado" para cada género. Sus testimonios fueron grabados y recogidos textualmente en cada guía y concentrados, según procediera, en 3 categorías de análisis: estereotipos masculinos, estereotipos femeninos y violencia. Resultados: Las mujeres entrevistadas reconocen la violencia masculina, los modelos diferenciados por género y las desigualdades sociales en detrimento de las mujeres pero lo consideran producto de las diferencias biológicas y del aprendizaje familiar materno. No hay claridad en las repercusiones de la violencia, en la salud de las mujeres salvo la violencia física. No ubican el maltrato como violación a sus derechos humanos. Hay en ellas el pensamiento mágico de que puedan manejar la violencia. Las relaciones sexuales conyugales forzadas son vistas como parte "natural" de la biología masculina por lo que requieren ser satisfechas por las mujeres para conservar su matrimonio. Diversas dependencias, religiosas y culturales, explican la tolerancia al maltrato. Conclusión: El considerar que la pertenencia a un género social constituye una categoría determinante en salud individual y colectiva, da a Enfermería otra dimensión realmente holística del cuidado.

Background: As almost throughout world in México is generally accepted that family violence is an ocute problem affecting a great number of people´s health; but it is also recognized that women are, at all ages, who more suffer int and that adult men are mostly the agressors. As an answer to de Mexican normativity within the NOM-190-SSA1-1999 which involves the nursing staff we draw-up as an. Objetctive: Identifying and describing family violence within a group of women (n=15), neighbars and users of the ENEO-UNAM´S Primary Health Care Community Center (CCOAPS) at San Luis Tlaxialtemalco, México (SLT). Procedure: We used a quialitative descriptive transversal research methodo disigned as an interview with previous informed const, within the CCOAPS physical settings, Social contruction models, based on gender perspective, were identified, and a guideline for the interview was elaborated and integrated with 11 items related to gender; the interview was conducted privately and confidentially assured, and it was audiotaped verbatim; the interviews were concentrated under 3 categories; male stereotypes, femenine stereotypes, and violence. Outcomes: Finding showed that women in the study sample acknowledge male violence, gender-differenciated models, and social inequities on detriment of women, but they consider them as a result of biological gender differences and family-maternal learning and don´t take violence as a violation to their human rigthts and dignity. Conclusion: Considering that being part of a social gender constitutes a determinant category on individual and collective health gives nursing another rerally holistic dimension of care.

Marker gene transfer into human haemopoietic cells using a herpesvirus saimiri-based vector.

Herpesvirus-based gene therapy vectors offer an attractive alternative to retroviral vectors because of their episomal nature and ability to accommodate large transgenes. Saimiriine herpesvirus 2 (HVS) is a prototypical gamma-2 herpesvirus that can latently infect numerous different cell types. A cosmid-generated HVS vector in which transforming genes have been deleted and the marker gene encoding enhanced green fluorescent protein (HVS-GFP) has been incorporated was evaluated for its potential to transduce CD34+ haemopoietic progenitors selected from cord blood. Expression of GFP could subsequently be readily detected in cells of the erythroid lineage in both CFU-GEMM assays and liquid differentiation cultures. These results confirm the potential of HVS as a candidate vector for gene therapy applications using primitive haemopoietic cells and suggest that it may be applicable to disorders affecting cells of the erythroid lineage.

What is the future for cord blood stem cells?

Stem and progenitor cells are present in cord blood at a high frequency making these cells a major target population for experimental and clinical studies. Over the past decade there has been considerable developments in cord blood research and transplantation but despite the rapid progress many problems remain. The initial hope that cord blood would be an alternative source of haemopoietic cells for transplantation has been tempered by the fact that there are insufficient cells in most cord blood collections to engraft an adult of average weight. In attempts to increase the cell number, a plethora of techniques for ex-vivo expansion have been developed.These techniques have also proved useful for gene therapy. As cord blood cells possess unique properties this allows them to be utilised as suitable vehicles for gene therapy and long-term engraftment of transduced cells has been achieved. Current work examining the nature of the stem cells present in this haematological source indicates that cord blood contains not only haemopoietic stem cells but also primitive non-haemopoietic cells with high proliferative and developmental potential. As attention focuses on stem cell biology and the controversies surrounding the potential use of embryonic stem cells in treatment of disease, the properties of stem cells from other sources including cord blood are being re-appraised. The purpose of this article is to review some of the current areas of work and highlight biological problems associated with the use of cord blood cells.

Cord blood G(0) CD34+ cells have a thousand-fold higher capacity for generating progenitors in vitro than G(1) CD34+ cells.

We examined the functional differences between G(0) and G(1) cord blood CD34+ cells for up to 24 weeks in serum-free suspension culture, containing Flt-3 ligand, thrombopoietin and stem cell factor. By week 24, there is more than a 1,000-fold difference in granulocyte, macrophage-colony-forming cells (GM-CFC) cumulative production between the two populations, with cultures initiated from G(0) demonstrating an amplification of 1.1 x 10(5)-1.8 x 10(6) of GM-CFC compared to 45-2.7 x 10(3) for the G(1) cells. Cells from the initial G(0) population are able to produce about 250-fold higher numbers of BFU-E than those from G(1) which translates to 3 x 10(3)-1.1 x 10(5)-fold expansion and 25-390-fold expansion for G(0) and G(1), respectively. This amplification of the progenitor cells is reflected in finding that a greater proportion of the progeny of the G(0) population are CD34+, resulting in a 600-fold expansion of CD34+ cells at week 8. As in other in vitro systems, total cell expansion is less discriminatory of stem cell behavior than progenitor cells, and there is no significant difference in total cell numbers between G(0) and G(1) cultures with a mean fold expansion of 2 x 10(7) at 24 weeks.

CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors.

Simple methods that separate progenitor cells of different hemopoietic lineages would facilitate studies on lineage commitment and differentiation. We used an antibody specific for the chemokine receptor CCR1 to examine mononuclear cells isolated from cord blood samples. When CD34(+) cells were separated into CD34(+)CCR1(+) and CD34(+)CCR1(-) cells and plated in colony-forming assays, the granulocyte/macrophage progenitors were found almost exclusively in the CD34(+)CCR1(+) cells. In contrast, the CD34(+)CCR1(-) cells contained the majority of the erythroid progenitors. There was a highly significant difference (P<0.002) in the total percentage distribution of both granulocyte-macrophage colony-forming cells and erythroid burst-forming units between the two populations. This is the first report of separation of erythroid progenitors from granulocyte/macrophage progenitors using a chemokine receptor antibody in cord blood samples. These results suggest that at the clonogenic progenitor cell stage the expression of CCR1 might be lineage-specific. This method should prove useful for studies on erythroid progenitor and granulocyte/macrophage differentiation.

NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor.

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.

Interest of cord blood stem cells.

Interest in cord blood stem cells was raised because of the possibility, now realised, of their use in clinical transplantation. The availability of only limited numbers of stem cells in cord blood compared to bone marrow or peripheral blood apheresis after cell mobilisation, led to experimental approaches that first aimed to characterise and then manipulate the stem cells present in cord blood. Their phenotypical and functional characteristics are not identical to those of stem cells in the bone marrow or those cells mobilised into the circulation. The cells selected for phenotype plus Go status show the higher capacity to generate progenitor cells in vitro and will offer the opportunity for mechanistic studies of stem cell self-renewal and proliferation. Another important field of exploration is to investigate the capacity of stem cells in cord blood for differentiation to tissues other than haemopoietic and to establish whether haemopoietic and non-haemopoietic lineages originate in truly multipotential cells or in cells coexisting in cord blood, which have already been limited to differentiation into specific tissue.

Transplantation potential of peripheral whole blood primed by VACOP-B chemotherapy plus filgrastim (r-metHuG-CSF) in patients with aggressive non-Hodgkin's lumphoma.

Large volumes of peripheral blood need to be processed to obtain sufficient stem cells for hematopoietic rescue after myeloablation, and more than one leukapheresis is necessary in most patients. We conceived the feasibility of harvesting sufficient numbers of hematopoietic cells from the whole blood, obtainable by venaepunctures, of patients treated with a standard dose chemotherapy regimen for high-grade non-Hodgkin's lymphoma. We evaluated the kinetics of mobilization, amount and quality of hematopoietic cells released into circulation during VACOB-B chemotherapy (which consists of a 12-week program), and G-CSF in 6 patients with aggressive non-Hodgkin's lymphoma. The median number of granulocyte-macrophage colony-forming cells (GM-CFC) x 10(3)/ml of blood (range), were 1.9 (0.3-8), and 1.16 (0.2-3.2) after the 7th and 11th weekly dose of drugs, respectively, showing an increase of 19- and 12-fold over patients' prechemotherapy values and of 53- and 33-fold over normal controls (p < 0.001). The median number of CD34+ cells x 10(3)/ml of blood (range), at the 7th and 11th cycle, was 135 (53.7-240.9) and 79.8 (69-173.5), respectively, showing an increase of 10- and 13-fold over patients prechemotherapy values (p < or = 0.04) and of 300- and 179-fold over normal controls (p < or = 0.001). Long-term culture initiating cells (LTC-IC) were released into circulation together with hematopoietic progenitors. We estimated that 1 liter of peripheral blood could yield on average 1.8 x 10(6)/kg CD34+ cells and 2 x 10(4)/kg GM-CFC with LTC-IC frequency comparable to a bone marrow harvest. These figures may be considered sufficient for hematopoietic rescue after myeloablation or hematopoietic support after high-dose chemotherapy.

Dendritic cells infected with recombinant fowlpox virus vectors are potent and long-acting stimulators of transgene-specific class I restricted T lymphocyte activity.

The identification of dendritic cells (DC) as the major antigen-presenting cell type of the immune system, combined with the development of procedures for their ex vivo culture, has opened possibilities for tumour immunotherapy based on the transfer of recombinant tumour antigens to DC. It is anticipated that the most effective type of response would be the stimulation of specific, MHC class I restricted cytotoxic T lymphocytes capable of recognising and destroying tumour cells. In order to make this approach possible, methods must be developed for the transfer of recombinant antigen to the DC in such a way that they will initiate an MHC class I restricted response. Here, we demonstrate that murine DC infected with a recombinant fowlpox virus (rFWPV) vector stimulate a powerful, MHC class I restricted response against a recombinant antigen. A rFWPV containing the OVA gene was constructed and used to infect the DC line DC2.4. The infected DC2.4 cells were found to stimulate the T-T cell hybridoma line RF33. 70, which responds specifically to the MHC class I restricted OVA peptide SIINFEKL. The stimulatory ability of the rFWPV-infected DC2.4 cells lasted for at least 72 h after infection and was eventually limited by proliferation of uninfected cells. By comparison, DC2.4 cells pulsed with synthetic SIINFEKL peptide stimulated RF33.70 well initially, but the stimulatory ability had declined to zero by 24 h after pulsing. FWPV infection of DC2.4 up-regulated MHC and costimulatory molecule expression. rFWPV was also found to infect both immature and mature human DC derived from cord blood CD34+ progenitors and express transgenes for up to 20 days after infection. We conclude that rFWPV shows promise as a vector for antigen gene transfer to DC in tumour immunotherapy protocols.

Multicentre European study comparing selection techniques for the isolation of CD34+ cells.

Primitive haemopoietic cells are required for studies in both the clinical and research fields and a number of systems have been developed to facilitate isolation of these haemopoietic cell populations. We have analysed the results from several European centres using positive selection of CD34+ cells from haemopoietic tissues (n = 110). Four selection techniques including immunoaffinity columns (Ceprate LC), immunomagnetic beads (Dynabeads, Baxter Isolex 50) and submicroscopic magnetic beads (MACS) were used and the selected CD34+ cells were assessed for purity, yield and enrichment of colony-forming cells (CFC). The mean purities for all samples ranged from 68.4-78.4% for MACS, 33.9-69.9% for Dynabeads, 46.9-66.8% for Ceprate LC and 43.2-65% for Baxter Isolex 50. Yields were variable with all techniques. On average CFC enrichment using the immunoaffinity columns was greater than that observed for the other systems. Some techniques appear to be problematic and may require further expertise to improve the results. Nevertheless, the study demonstrates that highly purified CD34+ cells can be isolated from various haemopoietic sources, though yield and CFC enrichment varies significantly depending on the technique selected. This extends our previous report indicating that not all selection methods generate similar results and that there are differences in the purity, number and colony-forming ability of the cells recovered.

Properties of peripheral blood and cord blood stem cells.

Mobilized peripheral blood and cord blood are used for transplantation in adults and children. Currently methods which assess the engraftment potential of these cells rely on nucleated cell count, clonogenic colony assays (GM-CFC) and CD34+ cell enumeration. However, data have accumulated which indicate that the cells responsible for short-term and long-term engraftment are different and may be identified by a variety of techniques, including immunophenotyping, in vitro and in vivo assays. There is also evidence that primitive cells in peripheral blood progenitor cell grafts and cord blood are heterogeneous, as cells with similar functional behaviour express different phenotypes. Despite intensive research, the isolation and identification of a homogeneous population of human stem cells is still elusive. Nevertheless, it is possible to obtain CD34+ subpopulations enriched in primitive cells with many of the properties expected of stem cells. Using these cell fractions, the cytokines that induce proliferation, amplification, differentiation and self-renewal are being defined in order to develop improved protocols for expansion of specific populations. From these studies a number of interesting facts have emerged. Certain growth factors frequently used for progenitor cell expansion and gene transduction studies also induce differentiation and impair long-term engraftment. Further, the cytokines required for progenitor cell expansion are probably different to those which favour expansion of the primitive cells, with both the cell cycle status of CD34+ cells as well as the implication of telomere shortening probably needing to be considered where ex vivo manipulation is contemplated.

CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors.

The AC133 antigen is a novel antigen selectively expressed on a subset of CD34+ cells in human fetal liver, bone marrow, and blood as demonstrated by flow cytometric analyses. In this study, we have further assessed the expression of AC133 on CD34+ cells in hemopoietic samples and found that there was a highly significant difference between normal bone marrow and cord blood versus aphereses (p <0.0001) but not between bone marrow and cord blood. Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133- cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 10(5) CD34+AC133- cells. The CD34+AC133+ population was also enriched (seven-fold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.

Expression of macrophage inflammatory protein-1 receptors in human CD34(+) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferon-gamma.

Macrophage inflammatory protein-1alpha (MIP-1alpha) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1alpha may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1alpha receptors on CD34(+) cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1alpha receptors on CD34(+) cells was analyzed by two-color flow cytometry using a biotinylated MIP-1alpha molecule. The mean percentage of LP CD34(+) cells expressing the MIP-1alpha receptors was 67.7 +/- 7.2% (mean +/- SEM; n = 22) as compared with 89.9 +/- 2.6% (n = 10) and 74.69 +/- 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1alpha receptor subtypes on LP CD34(+) cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34(+) cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34(+) cells for 24 to 36 hours in the presence of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) resulted in a significant increase in MIP-1alpha receptor expression. TNF-alpha induced MIP-1alpha receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1alpha receptor expression on hematopoietic cells.