Colin Robertson, Smile A While: Dentistry, History, Theater and Golf.

Many commentators argue that, until the 1980s, Brisbane and Queensland were cultural backwaters within the Australian context. However, with the hosting of the highly successful XII Commonwealth Games (1982) and World Expo 88 (1988) and with the development of the Queensland Cultural (1976-) and South Bank (1974-) Precincts, Brisbane and Queensland cast aside perennial apologism to acquire prominence, maturity, and self-respect. Within these national, state, and municipal settings, the theme, timing, and venue for the 24th Australian Dental Congress (1985) involved risk. Enter a Brisbane dental practitioner, Colin Robertson: a theater critic and a scriptwriter, who possessed competitive and entrepreneurial streaks, a vivid imagination, a steely resolve, and a love of golf. Robertson became an erudite, incisive, and prolific commentator, who penned much of the historical musical Smile A While (aka Smile Awhile) for the abovementioned congress. He contributed to an outstanding Dental Health Week (1980), served on the Australian Dental Association Queensland Branch Council (1981), its Fluoridation Committee (1973) and chaired both its Oral Health Education Committee (1981) and the Congressional Entertainment Division within the Congress Organizing Committee (1982-1985). Accordingly, the authors use historical methods to expose and explore a scarcely acknowledged narrative within dental history in Queensland.

James Meyrick Croker: A Model for Professional Behavior.

The rationale that underpins volunteering has long fascinated behavioral scientists. James Meyrick Croker's personal life, professional career and community engagement conform to the classic twentieth century model for professional behavior. Accordingly, the authors use historical methods of investigation to evaluate the influences on and the legacies from a remarkable contribution to the professions and the community. The narrative demonstrates elements of altruism, collaboration, conviction, compassion, drive, entrepreneurialism, familial and grammar school influence, leadership, pragmatism and vision. Croker's professional and community service was multi-organizational. Concurrent demands on his time warranted discipline, energy and expertise. For the behavioral scientist, achievement, affiliation, nature and nurture appear relevant to the outcome. Available archives provide no evidence of ego-driven motivation. Leadership style was transformational not transactional. Major legacies to the national and state Australian Dental Associations are ADAQ Christensen House (1972-1980), the eventual financial stability for the Australian Dental Association Queensland Branch, formal dental assistant training, policies of the Australian and Queensland Councils of Professions, a notable Goddard Oration and the successful 24th Australian Dental Congress.

Tumor necrosis factor (TNF)-mediated activation of the p55 TNF receptor negatively regulates maintenance of cycling reconstituting human hematopoietic stem cells.

Hematopoietic stem cell (HSC) fate decisions between self-renewal and commitment toward differentiation are tightly regulated in vivo. Recent developments in HSC culture and improvements of human HSC assays have facilitated studies of these processes in vitro. Through such studies stimulatory cytokines critically involved in HSC maintenance in vivo have been demonstrated to also promote HSC self-renewing divisions in vitro. Evidence for negative regulators of HSC self-renewal is, however, lacking. Tumor necrosis factor (TNF), if overexpressed, has been implicated to mediate bone marrow suppression. However, whether and how TNF might affect the function of HSC with a combined myeloid and lymphoid reconstitution potential has not been investigated. In the present studies in vitro conditions recently demonstrated to promote HSC self-renewing divisions in vitro were used to study the effect of TNF on human HSCs capable of reconstituting myelopoiesis and lymphopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Although all cord blood and adult bone marrow CD34(+)CD38(-) cells were capable of undergoing cell divisions in the presence of TNF, cycling HSCs exposed to TNF in vitro and in vivo were severely compromised in their ability to reconstitute NOD-SCID mice and long-term cultures. The negative effect of TNF was not dependent on the Fas pathway, and a similar effect could be observed using a mutant TNF exclusively targeting the p55 TNF receptor. TNF did not appear to enhance apoptosis or affect cell-cycle distribution of cultured progenitors, but rather promoted myeloid differentiation. Thus, TNF might regulate HSC fate by promoting their differentiation rather than self-renewal.

Combination chemotherapy containing mitoguazone, ifosfamide, methotrexate, etoposide (MIME) and G-CSF efficiently mobilize peripheral blood progenitor cells in heavily pre-treated relapsed lymphoma patients.

In this study we explored whether a standard chemotherapy regimen consisting of mitoguazone, ifosfamide, methotrexate and etoposide (MIME) combined with 5 micrograms/kg or 10 micrograms/kg G-CSF was capable of mobilizing peripheral blood progenitor cells (PBPC) in lymphoma patients. Thirty-three patients with Hodgkin's disease (HD) and 108 patients with non-Hodgkin's lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3-40) of chemotherapy prior to mobilization. Eight of 141 patients failed to mobilize PBPC and bone marrow was harvested. In addition, 10 patients obtained a harvest of < 2.0 x 10(6) CD34+ cells/kg. More than 2.0 x 10(6) CD34+ cells/kg were achieved in all HD patients and in 83% of the NHL patients. Fifty-eight per cent of the patients harvested > or = 5 x 10(6) CD34+ cells/kg. Eleven per cent of the patients developed neutropenic fever during the mobilization and 3% had nadir platelet values below 20 x 10(9)/L. An inverse correlation was observed in high-grade NHL (H-NHL) patients between the number of chemotherapy cycles given before mobilization and yield of CD34+ cells. Such an association was not seen among patients with HD, transformed and low-grade NHL. When G-CSF 10 micrograms/kg was used in combination with MIME, this correlation was no longer seen in patients with H-NHL. There was also association between CD34+ cell yield and prior radiotherapy in patients with HD or transformed NHL or low-grade NHL. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF, can be successfully used to mobilize PBPC. Although no significant effect of increased dose of G-CSF was found, our data suggest that MIME/G-CSF 10 micrograms/kg should preferentially be used to mobilize PBPC in H-NHL patients pre-treated with > or = 12 cycles of chemotherapy, in irradiated HD patients and in all low-grade and transformed lymphomas.

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells.

Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL-3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13-7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G-CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro.

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.

Inhibition of stem cell factor-induced proliferation of primitive murine hematopoietic progenitor cells signaled through the 75-kilodalton tumor necrosis factor receptor. Regulation of c-kit and p53 expression.

TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated.

Tumor necrosis factor (TNF)-alpha directly inhibits human erythropoiesis in vitro: role of p55 and p75 TNF receptors.

Two tumor necrosis factor receptors (TNFRs) with molecular weights of 55 kD (TNFR-p55) and 75 kD (TNFR-p75) have recently been identified and cloned. In previous studies, TNFR-p55 has been shown to exclusively mediate bidirectional effects of TNF-alpha on committed bone marrow granulocyte-macrophage progenitor cells, whereas both TNFR-p55 and TNFR-p75 can mediate inhibition of primitive progenitors requiring multiple cytokines to proliferate. We show here that TNF-alpha potently and directly inhibits the in vitro growth of committed erythroid progenitor cells in response to multiple cytokine combinations, and that TNF-alpha-induced inhibition of burst-forming unit-erythroid colony formation is mainly mediated through TNFR-p55, although TNFR-p75-mediated inhibition could be observed on progenitors responsive to erythropoietin alone. Moreover, at low TNF-alpha concentrations (2 ng/mL), TNF-alpha stimulates interleukin-3-dependent in vitro growth of committed granulocyte-macrophage progenitor cells, whereas it potently inhibits erythroid progenitor cell proliferation, showing that one concentration of TNF-alpha can simultaneously and bidirectionally modulate interleukin-3-dependent growth of committed granulocyte-macrophage (stimulation) and erythroid progenitor cells (inhibition).

All-trans retinoic acid directly inhibits granulocyte colony-stimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells.

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte-macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)-induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.

Role of the 75-kDa tumor necrosis factor receptor: inhibition of early hematopoiesis.

Biological effects of tumor necrosis factor alpha (TNF-alpha) are mediated through two cell surface receptors, the 55-kDa TNF receptor and the 75-kDa TNF receptor. The present study investigated the relative roles of the two TNF receptors in normal hematopoiesis. Using agonists (antibodies) specific for the 55- and 75-kDa TNF receptors, we demonstrate differential roles of the two TNF receptors in hematopoiesis in that only the 55-kDa TNF receptor mediates antiproliferative effects of TNF-alpha on mature Lin- hematopoietic progenitor cells responding to granulocyte colony-stimulating factor or interleukin 3 alone. In contrast, the 75-kDa TNF receptor is essential in mediating inhibition of primitive Lin-Sca-1+ high-proliferative-potential colony-forming cells and inhibition of the total number of proliferative clones of individually cultured Lin-Sca-1+Rh123lo and Lin-Sca-1+Rh123hi cells.

Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells.

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte-macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

Direct synergistic effects of interleukin-7 on in vitro myelopoiesis of human CD34+ bone marrow progenitors.

Interleukin-7 (IL-7) is an important growth factor in B and T lymphopoiesis in mouse and human, whereas IL-7 has been regarded to lack proliferative effects on cells within the myeloid lineage. However, we have recently reported that IL-7 potently can enhance colony stimulating factor (CSF)-induced myelopoiesis from primitive murine hematopoietic progenitors, showing a novel role of IL-7 in early murine myelopoiesis. Using CD34+ human hematopoietic progenitor cells, we show here a similar role of IL-7 in human myelopoiesis, although interesting differences between the two species were found as well. Although purified recombinant human (rh)IL-7 alone did not induce any proliferation of CD34+ cells, IL-7 in a concentration-dependent manner enhanced the colony formation induced by all four CSFs up to threefold. Furthermore, stem cell factor (SCF)-induced granulocyte-macrophage (GM) colony formation was increased fourfold in the presence of IL-7. Single-cell cloning assays showed that these synergistic effects of IL-7 were directly mediated on the targeted progenitors, and that IL-7 increased the number, as well as the size of the colonies formed. Morphological examination showed that IL-7 affected the progeny developed from CD34+ cells stimulated by G-CSF or IL-3, increasing the number of CFU-M (colony forming unit-macrophage) and CFU-granulocyte-macrophage, whereas the number of CFU-granulocyte were unaltered.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well.

Bifunctional effects of tumor necrosis factor alpha (TNF alpha) on the growth of mature and primitive human hematopoietic progenitor cells: involvement of p55 and p75 TNF receptors.

Tumor necrosis factor alpha (TNF alpha) has previously been reported to have both inhibitory and stimulatory effects on hematopoietic progenitor cells. Specifically, TNF alpha has been proposed to stimulate early hematopoiesis in humans. In the present study we show that TNF alpha, in a dose-dependent fashion, can potently inhibit the growth of primitive high proliferative potential colony-forming cells (HPP-CFCs) stimulated by multiple cytokine combinations. Using agonistic antibodies to the p55 and p75 TNF receptors or TNF alpha mutants specific for either of the two TNF receptors, we show that both receptors can mediate this inhibition. In contrast, the potent stimulation of interleukin-3 (IL-3) plus granulocyte-macrophage colony-stimulating factor (GM-CSF) induced HPP-CFC colony formation observed at low concentrations of TNF alpha (2 ng/mL) was only a p55-mediated event. Moreover, the stimulatory effects of TNF alpha on GM-CSF or IL-3-induced colony formation, as well as the inhibition of G-CSF-induced colony growth, were also exclusively signaled through the p55 TNF receptor. Taken together, our results suggest that the inhibitory effects of TNF alpha on primitive bone marrow progenitor cells are mediated through both p55 and p75 TNF receptors, whereas the p55 receptor exclusively mediates the bidirectional effects on more mature, single factor-responsive bone marrow progenitor cells as well as stimulation of IL-3 plus GM-CSF-induced HPP-CFC colony growth.

All-trans- and 9-cis-retinoic acid: potent direct inhibitors of primitive murine hematopoietic progenitors in vitro.

Retinoic acid (RA) stimulates the clonal proliferation of mature bone marrow progenitor cells and inhibits the growth of leukemic progenitors, whereas its effects on normal primitive hematopoietic progenitors have not yet been investigated. This study investigated the ability of all-trans- and 9-cis-RA to modulate the proliferation and differentiation of murine Lin-Sca-1+ bone marrow progenitor cells. Both RA isoforms inhibited in a reversible and dose-dependent fashion, the proliferation of multi- but not single-factor responsive Lin-Sca-1+ progenitor cells. The 50% effective dose was 10 nM for both all-trans- and 9-cis-RA. Maximum inhibition was observed at 100-1,000 nM RA, resulting in a 50-75% reduction in the number of proliferative clones. Lin-Sca-1+ cells with high proliferative potential were preferentially inhibited by RA, resulting in a 80-100% inhibition depending on the hematopoietic growth factors stimulating their growth. The inhibitory effects of RA were directly mediated on the target cell, since the effects were observed at the single cell level. Furthermore, autocrine transforming growth factor beta (TGF-beta) production can probably not account for the observed inhibitory effects of RA, since a TGF-beta neutralizing antibody did not block RA-induced inhibition. Whereas RA, in general, is a differentiation-inducing agent, treatment of Lin-Sca-1+ progenitors resulted in the accumulation of an increased fraction of blasts and immature myeloid cells. Thus, RA inhibits the proliferation as well as differentiation of normal primitive hematopoietic progenitor cells.

TNF-alpha, the great imitator: role of p55 and p75 TNF receptors in hematopoiesis.

The clinical application of tumor necrosis factor-alpha (TNF-alpha) has so far been limited due to the severe adverse effects associated with its systemic use. Recently, two distinct TNF receptors with molecular weights of 55 kDa (TNFR55) and 75 kDa (TNFR75) have been cloned and characterized. The subsequent development of TNF-alpha mutants with selective activity on either TNFR55 or TNFR75 suggest that such mutants might maintain the therapeutic (anti-tumor) potential of wild type TNF-alpha, but exhibit reduced toxicity (proinflammatory effects). In the present article we discuss previous studies on the effects of TNF-alpha in in vitro and in vivo hematopoiesis. In addition, we summarize more recent data from our laboratory as well as others, elucidating the role of TNF-alpha as a direct bifunctional regulator of in vitro hematopoiesis. Specifically, TNF-alpha is a potent inhibitor of the clonal growth of primitive and committed murine and human bone marrow progenitors in combination with multiple cytokines, including granulocyte colony-stimulating factor (G-CSF), CSF-1, erythropoietin (Epo), stem cell factor (SCF), and flt3 ligand (FL). In contrast, TNF-alpha at low concentrations can synergistically and directly enhance the clonal growth of primitive and more mature human CD34+ bone marrow progenitors when combined with GM-CSF or interleukin (IL)-3. Thus, a critical determinant of whether TNF-alpha elicits a stimulatory or inhibitory effect on the in vitro growth of hematopoietic progenitors appears to be the specific growth factors with which it interacts, rather than the maturity of the targeted progenitor. Furthermore, we describe the involvement of the two TNF receptors in signaling in vitro hematopoietic effects of TNF-alpha. Whereas TNFR55 is involved in most observed responses to TNF-alpha, signaling of TNFR75 appears to be restricted to inhibitory effects on primitive progenitors. Finally, we discuss the complexity of direct and indirect actions of TNF-alpha in in vivo hematopoiesis, and the potential clinical applications of TNF-alpha or TNF mutants.

Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.

[Infection problems in the leukopenia phase after autologous bone marrow transplantation]. / Infeksjonsproblemer i leukopenifasen etter autolog beinmargstransplantasjon.

Between 1986 and 1991, 29 patients with malignant lymphomas were treated with total body irradiation and high-dose chemotherapy followed by autologous bone marrow transplantation at the Norwegian Radium Hospital. Owing to treatment-induced bone marrow toxicity, the leucocyte count in peripheral blood rapidly fell to zero. The aplastic phase lasted for 25 days (median). All patients experienced fever in the posttransplant period and needed broad-spectrum antibiotics. 12 patients had documented bacteremia, mostly with Gram-positive isolates. There were seven cases of documented focal infections, and antibiotic-associated colitis was seen in two cases. Other complications included bleedings and skin rashes. Four patients died in the post-transplant period from complications related to the cytotoxic treatment. We discuss guidelines for antimicrobial treatment of suspected and confirmed infections in neutropenic patients.

[Treatment of iliofemoral venous thrombosis]. / Behandling av iliofemoral venøs trombose.

In a series of 41 patients with iliofemoral venous thrombosis, 23 were treated with heparin only. Five patients received ultra high dose streptokinase without any detectable thrombolysis. 11 patients were treated according to the conventional dose regimen for streptokinase. Three of these obtained complete thrombolysis, and two partial thrombolysis. Two patients were treated primarily with thrombectomy, whereas five patients were referred to thrombectomy after streptokinase had failed. Operation resulted in complete recanalization in four cases, and partial recanalization in two cases. We recommend the conventional dose regimen for streptokinase as first-line therapy for iliofemoral venous thrombosis. Thrombectomy should be considered if phlegmasia cerulea dolens is present, or when streptokinase is contraindicated. Thrombectomy can be carried out equally well after streptokinase failure, if major symptoms last less than a week.