Mechanisms of glucose transport at the blood-brain barrier: an in vitro study.

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

Adenosine permeation of a dynamic in vitro blood-brain barrier inhibited by dipyridamole.

Adenosine is an inhibitory neuromodulator in the central nervous system and has been reported to have neuroprotective properties. Using a dynamic in vitro blood-brain barrier, we investigated the hypothesis that inhibition of adenosine transporters on the lumenal side of the blood-brain barrier may decrease the loss of adenosine from the brain. Our results indicate that lumenal administration of dipyridamole, a nucleoside transport inhibitor, can inhibit adenosine permeation from the extracapillary space into the lumen.

Characterization of monoclonal antibodies that recognize canine CD34.

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34- cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit-granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34- cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of

The expression and differentiation pattern of cell antigens and adhesion molecules on the nonadherent cell population in canine long-term marrow culture: a biphasic development of myeloid and lymphoid cells.

During maturation of normal hematopoietic progenitors, there appears to be differentiation-dependent expression of adhesion receptors, which may contribute to the homing and lodging of stem progenitor cells into the marrow and for the eventual release of mature effector cells from the marrow cavity. Using a model of long-term marrow culture, we studied the expression pattern of different lineage cell antigens and cell adhesion molecules on the nonadherent cells in canine long-term marrow culture. CD4+ cells became a major proportion of both the small and large cell subsets by day 8 of culture. Small CD4+ cells, the majority of which are negative for other T-cell antigens during the first 2 weeks of culture, express low levels of CD4 (CD4lo) and coexpress granulocytic and/or monocytic markers. These CD4lo cells have progenitor potential as measured by the long-term culture-initiating cell assay and differentiate into myeloid (first wave) and lymphoid (second wave) cells. The T cells, which appear in 2-week-old long-term marrow culture, respond to Con A but not to alloantigen. At the same time, most of the large cells are CD14+, CD11b+, DLA-DR+ and CD4lo+, while granulocytes are not observed. This phenotypic pattern closely resembles that found on myelomonocytic cells developing from fetal thymic and fetal liver CD34+ precursors in a model of human fetal thymic organ culture. The cell adhesion molecules--CD44, CD18, L-selectin, CD11a-c as well as VLAalpha4, dramatically increase from the first week of long-term marrow culture, while ICAM-1 and a new beta2 cell adhesion molecule, alpha dbeta2, increased slightly from the third week on. In the large ("monocytic") cell population, alpha dbeta2 was exclusively expressed by CD4+ cells. The differentiation pattern of T-cell antigens and adhesion molecules seen in the canine long-term marrow culture appear to mimic those required for developmental interactions between leukocytes and endothelial cells; that is, an early expression of L-selectin and CD44, followed by the integrins, and later on by ICAM-1 and alpha dbeta2. Our data support the view that in a model of canine long-term marrow culture hematopoietic precursors retain their intrinsic developmental potential.

Enkephalins in hematopoiesis.

Recent data support the view that neuropeptide mediators, in particular opioid peptides, participate in the control of hematopoiesis. The main arguments are: neuropeptides modulate the functions of lymphoid cells, macrophages and mature granulocytes; they control cell proliferation and differentiation in many tissues, particularly during embryogenesis; lymphoid cells, macrophages, polymorphonuclear granulocytes and bone marrow stromal elements express neuropeptide receptors; bone marrow cells produce opioid-like neuropeptides; the CD10/CALLA marker of lymphoid, myeloid and marrow stromal cells is an enzyme, endopeptidase, which cleaves- and thus activates/inactivates-opioid and other neuropeptides. We have shown that opioid peptides enkephalins, opioid antagonist naloxone, and the inhibitor of enkephalin-degrading endopeptidase, thiorphan, modulate the proliferation and differentiation of hematopoietic cells in clonal and long-term cultures of mouse bone marrow. The effects partly depended on the presence of the accessory hematopoietic elements, and followed a circadian pattern. The dose-responses were irregular, showed strain-dependent and individual variations, and apparently reflected the state of the activity of target cells, cellular interactions and simultaneous signals by other mediators. The enkephalins were shown to bind to specific (opioid) receptors on the target cells, and their signals to be transmitted to the cell interior by a cascade of secondary messengers including diacyl-glycerol (DAG), protein-kinase C (PKC) and Ca++ ions. Neuropeptide regulation of hematopoiesis might belong to a complex immuno-neuroendocrine network including melatonin.

Canine CD34: cloning of the cDNA and evaluation of an antiserum to recombinant protein.

Increasingly, enriched populations of hematopoietic progenitors are used in experimental and clinical transplantation studies. The separation of progenitors is based on the expression of CD34, a marker preferentially expressed on progenitor cells. The dog model has been important for preclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To identify and isolate canine hematopoietic progenitors, we have cloned a cDNA encoding a CD34 homologue from a canine myelomonocytic leukemia cell line, ML2. The CD34 homologue cDNA predicts an amino acid sequence that is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C-terminal end of the extracellular domain, but shows considerable divergence from these sequences at the amino-terminal end of the protein. In Western blotting studies, canine CD34 homologue (caCD34) appears to be a heavily and variably glycosylated protein with a molecular weight of approximately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the expression of caCD34 protein, the extracellular domain of caCD34 was expressed as an Ig fusion protein and used as an immunogen to generate a rabbit polyclonal antiserum. The antiserum reacted against the fusion protein, against vascular endothelium, and with three leukemic cell lines. Approximately 1% of canine bone marrow cells stained brightly with antibodies to caCD34 and this population was 25- to 50-fold enriched for colony-forming units-granulocyte-macrophage as compared to unfractionated marrow mononuclear cells. These findings suggest that the canine CD34 homologue is expressed on bone marrow progenitor cells and, thus, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoiesis and stem cell transplantation.

Allogeneic transplant of canine peripheral blood stem cells mobilized by recombinant canine hematopoietic growth factors.

We have studied graft-versus-host disease (GVHD) after transplantation of allogeneic peripheral blood stem cells (PBSC) mobilized by either recombinant canine granulocyte colony-stimulating factor (rcG-CSF) alone or combined with stem cell factor (rcSCF). These studies were prompted by the observation of extremely rapid and sustained engraftment of growth factor-mobilized PBSC in the autologous setting using genetically marked cells and changes in function of T lymphocytes from donors that had undergone mobilization. Specifically, lymphocytes from growth factor-treated donors were hyporesponsive in mixed leukocyte culture and in response to Con A, raising hopes that GVHD in dogs given growth factor mobilized allogenic PBSC might be altered in a beneficial way. Eighteen dogs were given a median of 17.1 x 10(8) PBSC/kg from littermate donors after 920 cGy of total body irradiation without postgrafting immunosuppression. Donors were either genotypically DLA-identical (n = 9) or DLA-haploidentical (n = 9). The median number of colony-forming unit-granulocyte macrophage (CFU-GM) infused was 27 x 10(4)/kg, and the number of CD34+ cells in the transplant was on the order of 4.6 x 10(6)/kg. The dogs received a median of 52.8 x 10(7) CD4 cells/kg and 13.7 X 10(7) CD8 cells/kg. All 18 dogs had prompt hematopoietic engraftment of donor cells as assessed by chimerism studies using variable number tandem repeat, as well as cytogenetic markers. Three of the nine dogs given grafts from DLA-identical littermates had fatal GVHD, five had transient GVHD, and one had no GVHD. All nine DLA-haploidentical recipients of PBSC developed fatal hyperacute GVHD. In conclusion, the expectation about rapid engraftment was fulfilled. However, incidence and severity of acute GVHD after transplantation of mobilized PBSC were not different than previously reported for nonmobilized PBSC or marrow. This model will allow for further studies, including T-cell depletion to minimize GVHD without increasing graft rejection.

An antibody to CD44 enhances hematopoiesis in long-term marrow cultures.

Monoclonal antibody S5, which is specific for CD44, facilitates engraftment of major histocompatibility complex (MHC)-mismatched canine bone marrow (BM) when infused into recipient animals before total body irradiation (TBI) and marrow infusion. The precise mechanism by which S5 facilitates engraftment is not known. Previously published data in a murine long-term bone marrow culture (LTBMC) model with other anti-CD44 monoclonal antibodies (mAbs) demonstrated an abrogation of myelo- and lymphopoiesis in LTBMC. To address this issue in an in vitro setting, the effect of S5 on canine myelopoiesis in LTBMC was investigated. The data indicate that treatment of LTBMC with S5 causes an absolute increase in the number of progenitor cells as measured by a colony-forming unit-granulocyte/macrophage (CFU-GM) assay compared to cultures treated with control mAb (p < 0.0001). This effect was not observed with three other anti-CD44 mAbs used (Hermes-1, S3, and IM7). In addition, a concomitant decrease in the production of nonadherent cells was noted (p < 0.0001). These effects were evident in both autologous and allogeneic LTBMC systems. mAb S5 has been shown to enhance natural killer (NK) activity, and more recent data indicate the increased cytoxic effect to be partially mediated through CD18. Thus, a possible role for modulation of the CD18 antigen in LTBMC by S5 was assessed by addition of the anti-CD18 mAb 60.3. While the S5-induced reduction in total nonadherent cell production was not changed by addition of 60.3, the increase in progenitor cells stimulated by S5 was abrogated. These findings suggest a role for anti-CD44 antibody in changes in the marrow microenvironment that may be responsible for facilitation of donor engraftment and, at least in part, CD18 may be involved in this phenomenon. The establishment of this in vitro LTBMC model will enable the mechanism to be further dissected.

Suppressive effect of met-enkephalin on bone marrow cell proliferation in vitro shows circadian pattern and depends on the presence of adherent accessory cells.

The cellularity of femoral bone marrow and the content of the granulocyte-macrophage colony forming cells (GM-CFC) were followed in mice between 0600 h and 1800 h. The cellularity increased at the beginning of the light period, and the GM-CFC content at the end. Opioid pentapeptide methionine-enkephalin reduced the GM colony forming ability of the bone marrow cell suspensions in proportion to the GM-CFC content. Removal of the accessory cells reversed the enkephalin sensitivity pattern of the GM-CFC. The circadian variations have been ascribed to a neuroendocrine regulatory network involving the opioid peptides and affecting the bone marrow accessory cells. The work draws attention to the circadian activity pattern of hemoregulatory oligopeptides applicable as adjuvants to antineoplastic chemotherapy.

Naloxone behaves as opioid agonist/antagonist in clonal cultures of mouse bone marrow cells.

The opioid peptide methionine (Met)-enkephalin and the opioid-receptor blocking agent naloxone were added to unseparated or to progenitor-enriched cell suspensions of mouse bone marrow before assay in clonal cultures. Bone marrow samples harvested at 18:00 hours produced more granulocyte-macrophage (GM) colonies than the 06:00 hour samples, and were more sensitive to the proliferation inhibition by both agents. Additive inhibitory effects of naloxone with the enkephalin were occasionally seen. Thus, in this experimental system, naloxone could behave as an opioid agonist. However, there were examples of naloxone diminishing (blocking) the suppressive effect of the enkephalin, as a true opioid antagonist. Significant naloxone/enkephalin interactions occurred in opioid-sensitive (18:00 h) samples of unseparated bone marrow. The interactions were virtually absent in progenitor cell-enriched populations, indicating a significant role of accessory cells in opioidergic regulation of hematopoietic progenitors.

Enkephalinase-blocking agent thiorphan affects cell growth and differentiation in long term culture of mouse bone marrow.

Enkephalinase-blocking agent thiorphan was added to long-term cultures of mouse bone marrow cells at the time of culture initiation (time 0) or 2 weeks thereafter, when the stromal layer appears. Cellularity, cell morphology (in cytospin smears) and the yield of granulocyte-macrophage progenitor cells (GM-CFC assay in agar) were recorded. Low concentrations of thiorphan accelerated recovery of the cultures after an initial drop of the cell count. Expansion and maturation of the granulocytic lineage was promoted, with parallel decline of the GM-CFC yield. Thiorphan probably interfered with the activity of enkephalinase (endopeptidase 24.11) in the cultures. That enzyme is the CD10 surface marker (CALLA) of lymphoid, myeloid and stromal elements.

Naloxone interferes with granulocytopoiesis in long-term cultures of mouse bone marrow; buffering by the stromal layer.

Long-term cultures of mouse bone marrow cells were treated with naloxone, starting at the time of culture initiation or in the 2nd or 4th week of culture. Cell proliferation was suppressed and the ratio of immature and mature granulocytes to macrophages diminished by naloxone treatment. The effect depended on the timing of naloxone addition to the cultures and on its concentration, with a bell-shaped dose-response curve. High and low concentrations of naloxone (10(-4), 10(-6), 10(-14) M) interfered with hematopoiesis more strongly than the intermediate concentrations (10(-8) to 10(-12) M). Early cultures lacking the stromal layer were more sensitive to naloxone than the cultures with established stroma. The bell-shaped dose-response curve has been attributed to an interplay of specific (opioid-receptor-mediated) and nonspecific mechanisms. Opioidergic mechanisms apparently participate in the regulation of hematopoiesis.

Effect of enkephalins on bone marrow cells.

Mouse bone marrow cells were incubated with methionine- or leucine-enkephalin (10(-15)-10(-6) M) before seeding into soft agar cultures. In marrow samples harvested at different times, enkephalins decreased GM colony count on average by 30-40%. In individual experiments, however, the same concentration of enkephalins caused even stimulation, or at other times had effect. In view of the circadian periodicity of neuroendocrine functions and hematopoietic activity, the enkephalin effect on bone marrow cells was tested on marrow samples harvested at fixed time points (6 am, 6 pm), using enkephalin concentrations in the physiological range (10(-12)-10(-9) M). The seeding efficiency of the 6-pm cell population was on average 50% above that of the 6-am population. The 6-pm cell population was also more susceptible to the inhibitory effect of the enkephalins (35% inhibition) than the 6-am population (15% inhibition), and the variability in response was considerably reduced. With progenitor cell-enriched population, obtained by fluorescence-activated cell sorting (FACS) of 6-am bone marrow samples, in 3 out of 6 experiments Met- and Leu-enkephalin showed 30-35% inhibition of GM colony formation over a wide range of concentrations (10(-15)-10(-6)). In the other 3 experiments, suppression as well as stimulation or no alteration in colony count were observed. This variability probably reflected quality (purity) of the progenitor cell population, and may indicate that the enkephalins affected hematopoietic cells via a population of accessory cells.

[The origin and clonal proliferation of malignant altered cells in Hodgkin's disease]. / Porijeklo i klonska proliferacija maligno alteriranih stanica u morbus Hodgkin.

Monoclonal antibodies and techniques of molecular genetics have shown Hodgkin's disease to be a clonal neoplasm, and that Hodgkin's and Reed-Sternberg cells--malignant cells in this lymphoma--are either of B-lymphoid, T-lymphoid, or myelo-monocytic cell origin. Hodgkin and Reed-Sternberg cells have high multiplicative potential. Their scarcity in lymphoma tissue in relation to nonneoplastic cells has been attributed a) to prolonged cell-cycle, or b) to secretion of lymphokines by which malignant cells attract reactive cells and become "diluted" in formed lymphogranuloma. Different histological subtypes of Hodgkin's disease seem to be correlated with different origin of malignant cell clones. In lymphocytic depletion, malignant cells are usually of B-lymphoid origin; in mixed cellularity and nodular sclerosis they are usually of T-lymphoid origin. In some cases, malignant cell clone is neither of B-lymphoid, neither of T-lymphoid origin; these cells might be "frozen" at a very low differentiation stage.