Polyphasic characterization of Xanthomonas axonopodis pv. allii associated with outbreaks of bacterial blight on three Allium species in the Mascarene archipelago.

Based on the number of new reports during the last two decades, bacterial blight of onion (Allium cepa) is considered an emerging disease. The causal agent, Xanthomonas axonopodis pv. allii, is pathogenic to several Allium species after inoculation, but outbreaks worldwide have been primarily reported on onion. We describe a unique epidemiological situation in Réunion Island, France, with concomitant outbreaks on three Allium species, onion, leek (A. porrum), and garlic (A. sativum). There was no host specialization within Allium spp. among strains associated with the three host species. Based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism, strains associated with these outbreaks in Réunion Island were highly related genetically to strains isolated from diseased plant samples and contaminated seed lots in the neighboring island of Mauritius, where the disease has occurred since 1984. All AFLP haplotypes were identified as X. axonopodis pv. allii based on polymerase chain reaction analysis using specific primers, biochemical tests, and/or pathogenicity tests. Two genetically related groups of strains (A and B) that can be distinguished by AFLP, differential utilization of three carbon sources, and xanthomonadin pigment production were detected initially after establishment of the pathogen. In less than 10 years after the establishment of the pathogen there was nearly an extinction of group A strains in Réunion Island, suggesting differences in fitness between strains in the two groups.

Transplantation of a fetus with paternal Thy-1(+)CD34(+)cells for chronic granulomatous disease.

A fetus diagnosed with X-linked chronic granulomatous disease was transplanted with Thy-1(+)CD34(+) cells of paternal origin. The transplant was performed at 14 weeks gestation by ultrasound guided injection into the peritoneal cavity. The fetus was delivered at 38 weeks gestation after an otherwise uneventful pregnancy. Umbilical cord blood was collected and used to determine the level of peripheral blood chimerism as well as levels of functional engrafted cells. Flow cytometry was used to detect donor leukocytes identified as HLA-A2(-)B7(+) cells, whereas recipient cells were identified as HLA-A2(+)B7(-) cells. No evidence of donor cell engraftment above a level of 0.01% was found. PCR was used to detect HLA-DRB1*15(+) donor cells among the recipient's HLA-DRB1*15(-) cells, but no engraftment was seen with a sensitivity of 1:1000. The presence of functional, donor-derived neutrophils was assessed by flow cytometry using two different fluorescent dyes that measure reactive oxygen species generated by the phagocyte NADPH oxidase. No evidence of paternal-derived functional neutrophils above a level of 0.15% was observed. Peripheral blood and bone marrow samples were collected at 6 months of age. Neither sample showed engraftment by HLA typing using both flow cytometry and PCR. Functional phagocytes were also not observed. Furthermore, no indication of immunological tolerance specific for the donor cells was indicated by a mixed lymphocyte reaction assay performed at 6 months of age. While there appears to be no engraftment of the donor stem cells, the transplant caused no harm to the fetus and the child was healthy at 6 months of age. Analyses of fetal tissues, obtained from elective abortions, revealed that CD3(+) T cells and CD56(+)CD3(-) NK cells are present in the liver at 8 weeks gestation and in the blood by 9 weeks gestation. The presence of these lymphocytes may contribute to the lack of donor cell engraftment in the human fetus.

The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation.

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells.

Differential effects of interleukin-3, interleukin-7, interleukin 15, and granulocyte-macrophage colony-stimulating factor in the generation of natural killer and B cells from primitive human fetal liver progenitors.

The regulatory roles of a number of early-acting growth factors on the generation of natural killer (NK) cells and B cells from primitive progenitors were studied. Experiments focused on the contributions of granulocyte-macrophage colony-stimulates factor (GM-CSF) and interleukin-3 (IL-3) to the regulation of the early events of lymphopoiesis.Two progenitor populations isolated from human fetal liver were studied, CD38(-)CD34(++)lineage(-) (Lin(-)) cells (candidate hematopoietic stem cells [HSCs]) and the more mature CD38(+)CD34(++)Lin(-) cells. The effects of different cytokines on the generation of CD56(+)CD3(-) NK cells and CD19(+) B cells were studied in serum-deprived cultures in the absence of stroma.NK cells generated in vitro were able to kill NK-sensitive target cells, expressed NK-associated marker CD161 (NKR-P1A), but exhibited little or no expression of CD2, CD8, CD16, CD94/NKG2A, or killer cell inhibitory receptors (KIRs). Among the cytokine combinations tested, kit ligand (KL) and IL-15 provided the best conditions for generating CD56(+) NK cells from CD38(+)CD34(++)Lin(-) cells. However, either flk-2/flt3 ligand (FL), GM-CSF, IL-3, or IL-7 could partially substitute KL. All of these cytokines also supported the growth of NK-cell progenitors from candidate HSC, with the combination of IL-15, KL, GM-CSF, and FL generating the greatest number of CD56(+) cells. B cells were generated from both progenitor populations in response to the combined effects of KL, FL, and IL-7. Both B and NK cells were generated with the further addition of IL-15 to these cultures. The in vitro generated B cells were CD10(+), CD19(+), HLA-DR(+), HLA-DQ(+), and some were CD20(+), but no cytoplasmic or surface immunoglobulin M expression was observed. In contrast with NK lymphopoiesis, GM-CSF, IL-3, and IL-15 had no effect on the generation of B cells from CD38(-)CD34(++)Lin(-) cells, and GM-CSF inhibited B-cell generation from CD38(+)CD34(++)Lin(-) progenitors. These findings indicate a differential regulation of NK and B lymphopoiesis beginning in the early stages of hematopoiesis as exemplified by the distinctive roles of IL-7, IL-15, GM-CSF, and IL-3.

Early progenitor cells from human mobilized peripheral blood express low levels of the flt3 receptor, but exhibit various biological responses to flt3-L.

The biological effects of flt3-L, and the expression of its tyrosine kinase receptor (flt3, CD135) were investigated on the immature subsets of human circulating peripheral blood progenitors obtained from cancer patients or normal volunteer donors, after mobilization with rhG-CSF or chemotherapy. flt3 was expressed at low levels, and its expression increased concomitantly with expression of CD38 within the CD34+ cell population. Despite this low-level expression, flt3-L exerted synergistic effects with a combination of c-kit ligand, IL-3, IL-6, GM-CSF and G-CSF, mainly to induce proliferation of CD34+/CD38- cells. In addition, flt3-L increased the detection of HPP-CFC, both immediately after cell selection, and after 7 and 14 d of cultures. We conclude that flt3-L is active on circulating early mobilized haemopoietic progenitors, despite the low- level expression of its receptor.

Administration of Flk2/Flt3 ligand induces expansion of human high-proliferative potential colony-forming cells in the SCID-hu mouse.

The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells. Furthermore, higher percentages of CD33+ CD15- cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies.

Ex vivo manipulations alter the reconstitution potential of mobilized human CD34+ peripheral blood progenitors.

The phenotype and functions of CD34+ cells isolated from peripheral blood (PB) of steady-state healthy volunteers (ssPB-CD34), and of patients or healthy volunteers after mobilization (mPB-CD34) were investigated. ssPB-CD34+ cells contain a lymphoid cell population that co-express T or B cell markers, while mPB-CD34+ cells lack this population. After 5-day culture, significantly higher levels of expansion in cell, CD34+ cell, and HPP-CFC numbers were induced in ssPB-CD34+ cells, as compared to mPB-CD34+ cells. Hematopoietic reconstitution potential of these ex vivo manipulated CD34+ PBPC was evaluated in SCID-hu mice. It was found that ssPB-CD34+ cells retained the potential to reconstitute human bone marrow (BM), as well as thymus implanted in SCID animals. In contrast, only very low levels of reconstitution were detected in human hematopoietic tissues injected with cultured mPB-CD34+ cells. Reconstitution was restricted to myeloid cells, and no B cell reconstitution in bone marrow, or T cell reconstitution in thymus was achieved by these cells. The loss of B cell reconstitution potential of mPB-CD34+ cells was shown to be induced in a time-dependent manner during culture. These results indicate that mPB-CD34+ cells have different phenotypic and functional properties from ssPB-CD34+ cells. This may affect the efficacy of cell and gene therapy with mobilized PBPC.

Preferential photoinactivation of leukemia cells by aluminum phthalocyanine.

The efficacy of chloroaluminum phthalocyanine (AlPc) for photodynamic therapy (PDT) has been evaluated in vitro on acute myeloid leukemia (AML) cells, normal peripheral blood leukocytes (PBL) and mobilized peripheral blood stem cells (mPBSC). The selectivity of the treatment has been evaluated by mixing PBL and TF-1, an erythroleukemic cell line. Upon photoradiation, this photosensitizer leads to a significant and preferential photokilling of leukemia cells in comparison to normal cells. The use of stimulated lymphocytes in PBL/TF-1 mixtures instead of resting cells also leads to a preferential killing towards TF-1 although activated PBL are more affected than resting PBL. The analysis of AlPc intracellular emission by flow cytometry shows that the uptake of the dye by leukemia cells is faster. This good efficacy towards AML and the observed lower phototoxicity towards normal cells (PBL, normal progenitors) suggest that this phthalocyanine is a potential bone marrow purging agent.

Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver.

Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.

The characterization, molecular cloning, and expression of a novel hematopoietic cell antigen from CD34+ human bone marrow cells.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

Early ontogeny of the human marrow from long bones: an immunohistochemical study of hematopoiesis and its microenvironment.

We examined long bones from 42 human embryos and fetuses whose gestational ages ranged from 6 to 28 weeks. Bone rudiment sections were stained using a panel of monoclonal antibodies directed at antigens expressed by hematopoietic cells, endothelial cells, smooth muscle cells, fibro-blasts, and stromal cells, to describe the events preceding and accompanying the onset of hematopoiesis in the diaphyseal region. Five distinct stages were identified. Stage I (6.6 to 8.5 gestational weeks [gw]) was that of entirely cartilaginous rudiments: chondrocytes were dilated and capillaries with CD34+ endothelial cells were observed in the perichondral limb mesenchyme. At stage II (8.5-9 gw) chondrolysis was actively proceeding; numerous CD68+ cells were observed, interspersed within the marrow cavity. Stage III (9 to 10.5 gw) was characterized by the development of the vascular bed in the absence of detectable hematopoiesis. At mid-diaphysis, specific structures that we named primary logettes were discernible; they consisted of small chambers of connective tissue, framed by a loose network of CD45-negative cells organized around an arteriole and limited from the surrounding sinus by a clearcut lining of CD34+ endothelial cells flanked on their abluminal side by alpha SM actin+ myoid cells. Stage IV (10.5-15 gw) was characterized by the onset of hematopoiesis. Hematopoietic cells were found exclusively in the primary logettes that had considerably increased in size. Logettes filled with hematopoietic cells were immersed within large and almost empty vascular sinuses. Logettes were attached by a short pedicle to connective tissue adjacent to bone/cartilage remaining formations; this tissue contained very rare hematopoietic cells. Logettes were few, usually less than 10 per long bone, and found solely in the diaphyseal area. Most hematopoietic cells found inside logettes were CD15+ myelocytes; rarely seen were glycophorin A+ immature erythroblasts and CD34+ nonendothelial cells. Hematopoietic cells within the logettes were in contact with alpha SM actin+ myoid cells and flattened endothelial-like (although consistently CD34-negative), aligned cells limiting small capillary lumina. Stage V (16 gw onward) was that of final organization of the long bones with areas of fully calcified bone and areas of dense hematopoiesis where logettes were no longer visible. This study shows three major features of incipient long bone hematopoiesis: 1) absence of CD34+ hematopoietic precursors before the onset of hematopoiesis and extreme rarity of those in the emerging blood-forming marrow, 2) predominance of granulopoiesis, and 3) exclusive development in specific structures organized by vascular cells. This study also suggests that CD68+ cells are instrumental in the chondrolysis process while vascular cells (endothelial and myoid cells) may be the critical microenvironment at the onset of hematopoiesis.

Phenotypic, molecular, and functional characterization of human peripheral blood CD34+/THY1+ cells.

A subset of mobilized CD34+ cells present in patient aphereses expresses Thy1 (CDw90). This population contains most long-term culture initiating cells, as assayed with a murine stromal cell line. It also contains a significant proportion of colony-forming unit granulocyte macrophage, but very few burst-forming unit erythroid. The limited differentiation towards the erythroid lineage is further confirmed by the absence of GATA-1 mRNA in the CD34+/Thy1+ subset, and by the low level of c-kit expression. The CD34+/Thy1+ subset appears phenotypically and functionally heterogeneous, a finding consistent with its high representation, compared to phenotypes such as CD34+/CD38-. Therefore, while at least some of CD34+/Thy1+ cells may be infectable by retroviral vectors, as shown by the presence of a transcript for the receptor for murine amphotropic retroviruses, the use of this selection strategy to specifically target human stem cells appears questionable.

[Early ontogeny of the human hematopoietic system]. / Ontogénèse précoce du système hématopoïétique humain.

Immunohistochemistry was used to detect markers of the vascular, stromal and hematopoietic cell compartments in the human embryo and early fetus, from 3 to 15 weeks of gestation. CD34 expression was consistently observed at the surface of vascular endothelial cells from off earliest stages tested, at the single exception of embryonic liver blood vessels. Yolk sac hematopoiesis was very transient and limited to primitive erythropoiesis. Clusters of erythroblasts, monocytes and granulocytes appeared from 4 to 5 weeks of gestation in the liver rudiment. The early development of the bone marrow was marked by the rapid invasion, at 8 weeks, of long bone cartilaginous rudiments by CD68+ osteoclast precursors, CD34+ endothelial cells and by preosteoblasts, leading to the development of large vascular sinuses between ossifying trabeculae. Endogenous erythro- and granulopoiesis developed from week 11 in primary logettes always organized around an arteriole, in a loose stromal mesenchymal network established between the media of these arterioles and the sinusal endothelium. Round, hematopoietic CD34+ cells were seen occasionally in yolk sac blood vessels. In the liver they were rare and intermingled as single cells in the hepatocyte cords; strikingly, CD34+ hematopoietic cells could seldom be detected in the developing bone marrow. In contrast, compact clusters of non-endothelial, round CD34+ CD45+ hematopoietic cells were detected, during the 5th week of development, in close association with the ventral wall of the dorsal aorta. These cells exhibit phenotypic and functional characteristics of very primitive hematopoietic progenitors. This observation is in striking correlation with the evidence accumulated in animal models that stem cells for the late embryonic and adult hematopoietic systems develop inside the embryo per se, in the vicinity of the dorsal aorta. We thus suggest that these aorta-associated CD34+ cells, that exhibit an anatomic localization similar to that of the intraembryonic stem cells identified in the avian and murine embryo, are the real stem of human hematopoiesis.