Quantitative aspects of granulocytic progenitor cell (CFUc) mobilization from extravascular sites in dogs using dextran sulphate (DS)

The relationship between the increase in the blood CFUc concentration after intravenous injection of dextran sulphate (DS) and the pre-existing levels of spontaneously circulating CFUc was studied in dogs. After 15 mg DS/kg body weight the CFUc numbers per ml blood rose by a factor of 3.7 over the pre-injection values, from 78 +/- 11 (SEM) to 359 +/- 50, in normal dogs, and increased by a factor of 3.9 in 0.84-Gy-r-irradiated animals which had a reduced initial CFUc concentration per ml, from 35 +/- 8 to 116 +/- 43. The injection of 20 mg DS/kg body weight into unirradiated dogs caused an increase, by a factor of 11.5, of the pre-injection CFUc concentration, from 101 +/- 20 to 921 +/- 106. On the basis of the mobilization curves for individual dogs, a significant correlation was found between the normal blood CFUc value and the number of CFUc mobilized by DS for both dose levels. From the descending part of the mobilization curves obtained after 15 mg DS/kg body weight, kinetic parameters of canine circulating CFUc were derived. The mean blood transit time (t) was 1.4 +/- 0.5 hr and the half time (T/2) was 1.0 +/- 0.4 hr.     

Translating preclinical findings of (endothelial) progenitor cell mobilization into the clinic; from bedside to bench and back

It is generally accepted that angiogenesis plays a major role in tumor growth and numerous targeting agents directed against angiogenesis pathways have been developed and approved for clinical use. In the past years the concept of angiogenesis has developed into a multi-faceted process in which, besides local activation and division of endothelial cells, bone marrow derived progenitor cells (BMDPCs) contribute to neovascularization. A multitude of preclinical and clinical data indicates that the release of BMDPCs influences the response to certain anti-cancer modalities. In this review we provide an overview of all the preclinical and clinical studies contributing to this hypothesis and translate these findings to the clinic by pointing out the clinical implications these findings might have. The recent insight in the mechanism of a systemic host response, in response to various treatment modalities has shed new light on the mechanism of tumor regrowth, early recurrence and metastasis formation during or after treatment. This provides various new targets for therapy which can be used to improve conventional chemotherapy. Furthermore it provides a potential explanation why bevacizumab selectively enhances the effectiveness of only certain types of chemotherapy.     

Description of a suicide technique in vitro for granulocytic stem cells (CFUc) by hydroxyurea on normal human bone marrow

It has been possible, by studying 15 normal human bone marrow samples, to develop a technique for the suicide effect in vitro by hydroxyurea (H.U.), applied to human granulocytic stem cells (CFUc). This study suggests a contact time of medullary cells from 60 to 80 minutes in the presence of a hydroxyurea concentration 6 mM per 10(6) cells per ml in vitro. Preliminary results concerning the clusters are discussed.     

Sedimentation velocity characterisation of the cell cycle of granulocytic progenitor cells in monkey hemopoietic tissue

Sedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H³TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H³TdR exposure indicates that 55% of total CFC's in adult marrow are in G₀ or G₁ with a volume of 410 μ³, 42% are in S phase and of volume 450–950 μ³, and the remainder are in G₂ and mitosis with a volume of between 600–950 μ³. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G₂ component. H³TdR exposure confirmed the non-cycling status of these fetal progenitor cells.     

炎症和氧化应激对内皮祖细胞动员及其功能的影响

内皮祖细胞对于维持血管内皮完整性和血管稳态具有重要作用.增强EPC的数量和功能可使心血管疾病患者获益.炎症、氧化应激对内皮祖细胞动员及其功能发挥具有重要影响,本文着重综述炎症和氧化应激对内皮祖细胞动员的调控,并探讨增进内皮祖细胞数量和功能的相关治疗策略.     

Cytokine-dependent granulocytic differentiation. Regulation of proliferative and differentiative responses in a murine progenitor cell line

Human granulocyte colony stimulating factor (G-CSF) can support the survival and short term proliferation of the interleukin 3 (IL 3)-dependent diploid murine hemopoietic progenitor cell line 32D C13. After 8 days in the presence of 30 U/ml of G-CSF and in the absence of IL 3, the great majority of 32D C13 cells becomes positive for myeloperoxidase (a marker that appears at the promyelocytic stage of the granulocytic lineage) and progressively differentiates into lactoferrin-containing neutrophilic granulocytes. Myeloperoxidase mRNA rapidly increases after 24 to 48 hr of treatment with G-CSF, peaks at day 6 and is no longer detectable at day 9 and 12, paralleling the appearance of myeloperoxidase-positive promyelocytes and myelocytes in the culture. After 12 days, 100% of the cells terminally differentiate, and clonogenic assays in IL 3-containing semisolid media indicate that the whole population has irreversibly lost proliferative capability. By using varying concentrations of both murine IL 3 and recombinant human G-CSF, the cultures develop an heterogeneous population of cells representing all the differentiation stages of the myeloid lineage, and the relative ratios of immature proliferating precursors and terminally differentiated cells present in the cultures can be modulated by modifying the concentrations of IL 3 or recombinant human G-CSF. Isobolic curves indicate that IL 3 and G-CSF have an antagonistic effect on the proliferation of 32D C13 cells. Thus, these cells represent a simplified in vitro model of normal granulocytic differentiation whose extent may be modulated completely in the presence of serum by two well-defined growth and differentiation factors: IL 3 and G-CSF.     

Quantitative aspects of harbour seal (Phoca vitulina) play

The play behaviour of harbour seals was analysed on videotape, which allowed the determination of rates of play per age class relative to the population of seals of the same age, and sex of the player in 190 episodes. The majority of the results of our earlier study were confirmed in that adult play was unusually common, and most play was solitary rather than social. However, the reason for the apparent predominance of juvenile play in our 1986 study was most likely because of a large number of juvenile seals present in the herd at that time. Similarly, mothers, pups and weaners proved to play at high rates when population size was taken into account. Adults' playing increased during the weaning/mating period, whereas juveniles and subadults played progressively less over time. Mothers and pups played more near weaning, after which time the youngsters played at a steady high rate, though never socially. Adult social play was only evident during the mating period. The play of males and females differed qualitatively and quantitatively.     

New aspects of the possible sites of ultrafiltration in annelids (oligochaeta)

Electron microscopic investigations of blood vessels were conducted to show sites of filtration such as podocytes or fenestrated endothelia. The endothelia of the blood vessels of Aelosoma hemprichi, Nais elinguis, Dero obtusa and Enchytraeus buchholzi consist of myoendothelial cells, chloragocytes and podocytes. The podocytes form large archs over a considerable area of the vessels. On the lumen side of the vessel there are several columnar processes which split into numerous small pedicels. The gaps between the adjacent pedicles are bridged by slit membranes. The podocytes are restricted to the front part of the ventral vessel. They are presumed to form a filtration surface. Furthermore, some parts of the ventral vessel are formed by a fenestrated endothelium, mainly in Enchytraeus buchholzi. In the vascular system of E. buchholzi two separate filtration sites were found. Additionally to the filtration site between ventral vessel and coelomic cavity a second filtration site was found in the front part of the body between blood sinus and coelomic cavity. In such areas the basement membrane is the only continuous layer between the blood vessel and the coelomic cavity. Its thickness is in the range of 40 nm. Possible filtration sites in the form of podocytes and irregular fenestrations could be localized at the border between the blood compartment and the coelomic compartment. It can be presumed that the primary urine may be formed by ultrafiltration of blood.     

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochromec oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and betahydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochromec oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochromec oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.     

Additive effect of endothelial progenitor cell mobilization and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs

Summary The methods of therapeutic angiogenesis include endothelial progenitor cell (EPC) mobilization with cytokines [e.g., granulocyte colony-stimulating factor (G-CSF)] and bone marrow mononuclear cell (BMMNC) transplantation. Combined angiogenic therapies may be superior to a single angiogenic therapy for the treatment of limb ischemia. Therefore, we investigated whether the angiogenic efficacy of a combination of two angiogenic strategies is superior to either strategy alone. One day after the surgical induction of hindlimb ischemia, mice were randomized to receive either no treatment, EPC mobilization with G-CSF administration, BMMNC transplantation using a fibrin matrix, or a combination of EPC mobilization with BMMNC transplantation using a fibrin matrix. EPC mobilization with G-CSF or BMMNC transplantation using a fibrin matrix significantly increased the microvessel density compared with no treatment. Importantly, a combination of EPC mobilization with BMMNC transplantation using a fibrin matrix further increased the densities of microvessels and BrdU-positive capillaries compared to either strategy alone. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression was higher in the EPC mobilization with G-CSF or BMMNC transplantation group than in the no treatment group. The combination therapy of EPC mobilization with G-CSF and BMMNC transplantation resulted in more extensive expression of bFGF and VEGF than the single therapy of either EPC mobilization with G-CSF treatment or BMMNC transplantation. This study demonstrates that the combination therapy of BMMNC transplantation and EPC mobilization potentiates the angiogenic efficacy of either single therapy in mouse limb ischemia models.     

Dose- and time-related quantitative and qualitative alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after total-body irradiation

The effects of single-dose total-body X irradiation (TBI) on the granulocyte/macrophage progenitor cell (GM-CFC) population in bone marrow and blood of dogs were studied for dose levels of 0.78 and 1.57 Gy up to 164 days after irradiation. The blood GM-CFC concentration per milliliter was depressed in the first 7 days in a dose-dependent fashion to 5-16% of normal after 0.78 Gy and to between 0.7 and 5% after 1.57 Gy. The bone marrow GM-CFC concentration per 10(5) mononuclear cells, on the other hand, was initially reduced to about 45% of the average pre-irradiation value after 0.78 Gy and to 23% after 1.57 Gy. The regeneration within the first 30 to 40 days after TBI of the blood granulocyte values and the repopulation of the bone marrow GM-CFC compartment was associated with both a dose-dependent increase in the S-phase fraction of the bone marrow GM-CFC and a dose-dependent increase in colony-stimulating activity (CSA) in the serum. The slow repopulation of circulating blood GM-CFC to about only 50% of normal even between days 157 and 164 after TBI could be related to a correspondingly delayed reconstitution of the mobilizable GM-CFC subpopulation in the bone marrow.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Generation of novel sequence tagged sites (STSs) from discrete chromosomal regions using Alu-PCR

Human DNA segments from discrete chromosomal regions were generated by utilizing Alu-element-based polymerase chain reaction (Alu-PCR) of an irradiation-fusion hybrid containing approximately 10 to 15 Mb of human DNA. Following cloning into a plasmid vector, a subset of the clones was used to generate sequence tagged sites (STSs) de novo. By means of a panel of hybrids containing portions of the human X chromosome, the STSs were shown to localize to two chromosomal regions, Xq24-Xq26 and Xcen-Xq13, reflecting the presence in the irradiation-fusion hybrid of two human chromosome fragments. These results demonstrate that high densities of STSs can be rapidly and efficiently generated from defined regions of the human genome using Alu-PCR.     

Igh restriction of the anti-alpha (1-3) dextran response: polyclonal B cell activators induce the synthesis of anti-alpha (1-3) dextran antibodies in lymphocytes from Igha mice only

The only strains of mice which are able to synthesize lambda 1-bearing antibodies in response to alpha (1-3) Dextran are those expressing the Igha allotypic haplotype or those having an Igh V region identical to Igha mice. The experiments reported here were designed to investigate whether the nonresponsiveness of mice which do not express the Igha haplotype is a consequence of an absence of a polyclonal B cell receptor for the alpha (1-3) Dextran TI-antigen. B cells of several mouse strains were stimulated with polyclonal B cell activators (PBA) known to either stimulate non-overlapping B cell subsets or to stimulate B cells at different stages of maturation, i.e., lipopolysaccharide, Nocardia delipidated cell mitogens and alloreactive T helper cells. Whereas all three PBA induced B cells from Igha mice to secrete lambda 1-bearing anti-alpha (1-3) antibodies, the PBA were incapable of inducing B cells from non-Igha mice to mount an anti-alpha (1-3) Dextran response. The data suggest that non-Igha mice lack a functional VH Dex gene for the lambda 1-bearing anti-alpha (1-3) Dextran response.     

Quantitative aspects of sperm transfer in Simulium decorum (Diptera: Simuliidae)

The mean number of sperm ejaculated into the spermatophore by the male black fly, Simulium decorum Walker, was 4048, as determined from 43 counts. The completeness and rate of sperm transfer were estimated by counts from the spermatophore and female spermatheca at selected times after copulation ended. The beginning of transfer occurred at the earliest about 4 min after copulation, but could be delayed until 30 min. Transfer rate was interpreted as constant at about 73 sperm/min and was usually completed by about 55 min after copulation. All spermatozoa appeared to be moved to the single spermatheca.     

The gastric epithelial progenitor cell niche and differentiation of the zymogenic (chief) cell lineage

In the mammalian gastrointestinal tract, the cell fate decisions that specify the development of multiple, diverse lineages are governed in large part by interactions of stem and early lineage progenitor cells with their microenvironment, or niche. Here, we show that the gastric parietal cell (PC) is a key cellular component of the previously undescribed niche for the gastric epithelial neck cell, the progenitor of the digestive enzyme secreting zymogenic (chief) cell (ZC). Genetic ablation of PCs led to failed patterning of the entire zymogenic lineage: progenitors showed premature expression of differentiated cell markers, and fully differentiated ZCs failed to develop. We developed a separate mouse model in which PCs localized not only to the progenitor niche, but also ectopically to the gastric unit base, which is normally occupied by terminally differentiated ZCs. Surprisingly, these mislocalized PCs did not maintain adjacent zymogenic lineage cells in the progenitor state, demonstrating that PCs, though necessary, are not sufficient to define the progenitor niche. We induced this PC mislocalization by knocking out the cytoskeleton-regulating gene Cd2ap in Mist1^−/− mice, which led to aberrant E-cadherin localization in ZCs, irregular ZC-ZC junctions, and disruption of the ZC monolayer by PCs. Thus, the characteristic histology of the gastric unit, with PCs in the middle and ZCs in the base, may depend on establishment of an ordered adherens junction network in ZCs as they migrate into the base.