Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages.

The transcription factor PU.1 is a hematopoietic-specific member of the ets family. Mice carrying a mutation in the PU.1 locus were generated by gene targeting. Homozygous mutant embryos died at a late gestational stage. Mutant embryos produced normal numbers of megakaryocytes and erythroid progenitors, but some showed an impairment of erythroblast maturation. An invariant consequence of the mutation was a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. Thus, the developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor.

The hunt for cancer-initiating cells: a history stemming from leukemia.

Conventional cancer therapies are plagued by disease relapses due to incomplete eradication of cancer-initiating cells. Evidence for cancer-initiating cells originally arose from studies in hematology and leukemia. Lessons learned from hematopoietic stem cells laid the bedrock for understanding how leukemic cells self-renew and remain in immature states. Decades later, leukemia-initiating cell techniques are now being applied to the field of solid tumors such as brain, breast, bone, colon, pancreas, lung and prostate cancer, with several cancer-initiating cell efforts led by hematologists. Different isolation techniques enriching for primitive cancer-initiating cells have been developed and are described in this review. Although the concept of cancer-initiating cells arose from studies in normal tissue stem cells, differences exist between neoplastic-initiating clones and their normal counterparts. Several efforts have uncovered aberrant molecular pathways and niche interactions unique to cancer-initiating cells. Efforts to exploit these pathways and interactions could ultimately lead to complete eradication of cancers.

Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI.

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.

Normal myeloid development requires both the glutamine-rich transactivation domain and the PEST region of transcription factor PU.1 but not the potent acidic transactivation domain.

Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore, PU.1(-/-) embryonic stem (ES) cells fail to differentiate into Mac-1(+) and F4/80(+) macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability of PU. 1(-/-) ES cells to differentiate into macrophages. In this study, we take advantage of our PU.1(-/-) ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1(-/-) ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

Extended time-lapse in vivo imaging of tibia bone marrow to visualize dynamic hematopoietic stem cell engraftment.

Homing, engraftment and proliferation of hematopoietic stem/progenitor cell (HSC/HPCs) are crucial steps required for success of a bone marrow transplant. Observation of these critical events is limited by the opaque nature of bone. Here we demonstrate how individual HSCs engraft in long bones by thinning one side of the tibia for direct and unbiased observation. Intravital imaging enabled detailed visualization of single Sca-1+, c-Kit+, Lineage- (SKL) cell migration to bone marrow niches and subsequent proliferation to reconstitute hematopoiesis. This longitudinal study allowed direct observation of dynamic HSC/HPC activities during engraftment in full color for up to 6 days in live recipients. Individual SKL cells, but not mature or committed progenitor cells, preferentially homed to a limited number of niches near highly vascularized endosteal regions, and clonally expanded. Engraftment of SKL cells in P-selectin and osteopontin knockout mice showed abnormal homing and expansion of SKL cells. CD150+, CD48- SKL populations initially engrafted in the central marrow region, utilizing only a subset of niches occupied by the parent SKL cells. Our study demonstrates that time-lapse imaging of tibia can be a valuable tool to understand the dynamic characteristics of functional HSC and niche components in various mouse models.

CT characteristics of a transplantable canine glioma model: preliminary kinetic analysis.

Experimental brain tumors can be produced in dogs through the intracerebral injection of 3 X 10(6) live tumor cells in either neonates or adult animals. Tumors are visible by computed tomography on day 8 postinjection. Most tumors appear as ring lesions with central lucencies and shaggy borders. By postinjection day 12, tumor volumes increase more than 10 times; the cell cycling time is about 1-3 days. The initial doubling time is about 1-2 days and corresponds to the in vitro doubling time of about 24 hr. The use of computed tomography to perform noninvasive kinetic analysis deserves further study. The transplantable canine glioma model would appear to be ideal for this purpose.

Spinal subarachnoid hematoma complicating lumbar puncture: diagnosis and management.

Two patients with altered hemostatic mechanisms developed spinal subarachnoid hemorrhage after difficult lumbar punctures. One patient had received anticoagulation therapy soon after lumbar puncture and the other had a low platelet count (63,000/mm3) at the time of lumbar puncture. In both patients a hematoma evolved, producing blockage of cerebrospinal fluid flow. Clinical manifestations consisted of severe back and radicular pain with sphincteric disturbances followed by rapidly developing severe paraparesis. Of the methods of radiographic evaluation that were used, including computed tomography (CT) without contrast enhancement, myelography, CT with intrathecally administered contrast medium, and magnetic resonance imaging, we found the best study to be myelography via lateral cervical puncture followed by CT. Unfortunately, diagnosis was delayed, and surgical evacuation of the hematomas did not substantially improve the patients' conditions. The salient clinical and radiographic features of this disorder and its pathophysiology are reviewed. Prompt recognition of these lesions is necessary so that surgical intervention may maximize chances of recovery.

Carotid endarterectomy complicated by vein patch rupture.

Saphenous vein patch angioplasty has been used to improve the results of carotid endarterectomy by decreasing the incidence of postoperative occlusion and recurrent stenosis. A rare but potentially lethal complication of this technique is aseptic necrosis and rupture of the vein patch during the postoperative period. We report three cases of this phenomenon and review an additional 13 cases from the literature. This event generally occurs without warning 2 to 7 days postoperatively and may result in death or stroke. At reoperation, the central portion of the vein patch is necrotic, without evidence of infection. Technical considerations in the harvesting and preparation of these grafts are reviewed, as are the physical parameters predisposing certain vein patches to rupture. Saphenous vein harvested from the ankle has been linked to every reported case. Small diameter veins in particular appear to carry a higher risk of rupture.

Transplantable canine glioma model for use in experimental neuro-oncology.

The need for a large animal tumor model in experimental neuro-oncology led us to re-evaluate and to modify the transplantable canine glioma of Wodinsky and Walker. Successive passages of the original tumor brei were made in purebred beagles, from beagle to mongrel, and between various mongrel strains until an intracerebral injection of 0.1 cc on Days 1 to 3 of life produced a 93% incidence of tumor take in all breeds. The mean survival was 13.5 +/- 1.9 days after injection (range, 10 to 19 days) in 10 litters. The tumor was invariably fatal and possessed many of the histological characteristics of human glioblastoma (i.e., capillary proliferation, pseudopallisading, frequent mitotic figures, and multinucleated giant cells). The animals were large enough to be scanned on the Pfizer 450 scanner, and the tumors were visualized in vivo as typical "ring" lesions after the injection of contrast agent. Intravital staining with Evans blue outlined the areas of contrast enhancement observed in the same tumors by computed tomography. The apparent defect in the blood-brain barrier could be explained in part by the absence of endothelial tight junctions on electron microscopy. Stability in the histology and activity of the tumor could be demonstrated after more than 14 months of storage at -70 degrees C. The transplantable canine glioma model has many advantages including low cost, reproducible morphology, a short survival time, and relative safety for the investigator. The large size of the animal preparation allows the use of complex surgical instrumentation and radiographic study, as well as repeated sampling of cerebrospinal and other fluids.

Type I fractures of the odontoid process: implications for atlanto-occipital instability. Case report.

Only four cases of Type I odontoid fracture have been previously described in the English literature. Most authors consider this lesion to be stable, although the mechanism(s) of injury has not been clearly elucidated. A case of Type I odontoid fracture in association with atlanto-occipital and atlantoaxial dislocation resulting in death is presented. The normal ligamentous anatomy is reviewed and proposed mechanisms for this injury are discussed. The radiographic features of all reported cases of this type are reviewed. It is proposed that the Type I odontoid fracture is a likely manifestation of atlanto-occipital instability and rarely occurs as an isolated or stable injury.

Level of benzimidazole resistance in a strain of Ostertagia circumcincta studied over several infections in lambs.

A benzimidazole-resistant strain of Ostertagia circumcincta (HFRO) was used experimentally to infect lambs. The level of resistance, measured by an egg hatch assay, was studied throughout each infection and also after treatment of the lambs with fenbendazole. The HFRO strain was highly resistant to benzimidazoles. There was day-to-day variation in the level of resistance throughout a single infection with a high level of resistance in the early part of the infection, around Day 27 post-inoculation of infective larvae, falling to a lower level later in the infection. Egg hatch assays on the 3 days immediately post-treatment with fenbendazole showed the resistance level was high then resistance fell to the pre-treatment level after 7 days. Selection for benzimidazole resistance using fenbendazole treatment at the normal dose rate of 5 mg kg-1 over five passages of the HFRO strain in lambs failed to increase the resistance level. Storage of larvae over a 5-month period at 4 degrees C, prior to infection of lambs, did not produce any alteration in the resistance level. The possible reasons for the variations in resistance found with the HFRO strain are discussed along with the implications for sheep parasite control and further development of benzimidazole resistance.

Fecundity of anthelmintic resistant adult Haemonchus contortus after exposure to ivermectin or benzimidazoles in vivo.

A strain of Haemonchus contortus resistant to the benzimidazoles, ivermectin and salicylanilides was used to study the effect of exposure to ivermectin or oxfendazole on the number of eggs produced by adult female parasites. After anthelmintic administration there was a reduction in the faecal egg count of the infected lambs. Ivermectin caused a reduction in the number of eggs within adult parasites for up to 72 hours but this returned to higher than pre-treatment levels at 168 hours after treatment. After oxfendazole treatment there was an increase in the number of eggs stored in the adult parasites at 24 hours and then a significant reduction in the number of eggs for over 168 hours. This suggested that both ivermectin and oxfendazole have a deleterious effect on the reproductive potential of female H contortus although the parasites are resistant to these drugs. These results confirm the importance of allowing up to 10 days to elapse between the time of administration of an anthelmintic and the collecting of faecal samples when using the faecal egg count reduction test to identify anthelmintic resistance, especially when using benzimidazoles. The more prolonged effect on fecundity produced by oxfendazole may reflect the antimitotic action of the benzimidazole anthelmintics.

Production of strains of the sheep parasite Ostertagia circumcincta by implantation of adult parasites into the abomasum of lambs.

The implantation of a single pair of adult parasites of Ostertagia circumcincta via the abomasa of four lambs resulted in the production of 19, 53, 393 and 425 viable larvae after faecal culture using faeces collected from day 6 to day 20 after implantation. Implantation of a single female parasite into two lambs failed to produce larvae.

Efficacy of ivermectin against Cooperia curticei infection in sheep.

Lambs were inoculated with a single dose of Cooperia curticei. Subcutaneous administration of ivermectin at a dosage of 200 micrograms/kg of body weight resulted in 61.1% and 90.4% anthelmintic efficacy, when measured at 7 and 14 days after treatment, respectively. In the treatment groups, parasites that remained were located more distally in the small intestine than those in the lambs in the control groups.

Effect of development of resistance to benzimidazoles, salicylanilides and ivermectin on the pathogenicity and survival of Haemonchus contortus.

Two groups of six lambs were infected either with an anthelmintic-susceptible strain of Haemonchus contortus or with a strain resistant to benzimidazoles, ivermectin and salicylanilides. The pathogenicity of the two strains of parasite was compared by monitoring the development of anaemia, changes in plasma proteins and abomasal damage in the two groups of lambs. There were no significant differences between the groups, suggesting that the development of resistance to several anthelmintics did not correlate with an increase in the pathogenicity of the parasites. Furthermore, the establishment rates and egg production of the susceptible and resistant parasites were similar. However, fewer of the eggs of the resistant parasites survived and developed at a variety of temperatures.

Clinical and pharmacological properties of ivermectin in rabbits and guinea pigs.

When 400 micrograms ivermectin/kg was administered subcutaneously to rabbits infected with the ear mite Psoroptes cuniculi it significantly reduced the clinical score, and when 500 micrograms ivermectin/kg was administered subcutaneously to guinea pigs with mange due to Trixacaurus caviae it resulted in a clinical cure. In rabbits a subcutaneous dose of 400 micrograms/kg produced high and sustained concentrations of ivermectin in the tissues and body fluids for at least 13 days and its rate of depletion from tissues was similar to that observed in sheep and rats. The mean (+/- sem) maximum concentration in plasma was 42.0 +/- 9.7 ng/ml 37.2 +/- 5.0 hours after administration and the area under the concentration-time curve was 3543 +/- 580 ng/ml hours. After the administration of 500 micrograms ivermectin/kg to guinea pigs orally, subcutaneously or topically the drug could be detected in the plasma only after subcutaneous administration. The mean concentration 72 hours after its administration to four guinea pigs was 0.7 +/- 0.3 ng/ml.

The distribution and some pharmacokinetic parameters of ivermectin in pigs.

Ivermectin was injected subcutaneously into five pigs at the usual dose rate of 300 micrograms/kg and found to distribute well to all tissues and body fluids which were sampled 24 h post-injection. Ivermectin was detected in the contents and mucus at all levels of the gastrointestinal tract. The drug was excreted in bile, with high concentrations of the drug in the intestines and faeces. High concentrations of ivermectin were measured in skin, ears and ear wax, suggesting that the drug should be effective in the treatment of ectoparasitic infestations, particularly ear mites. The high lipid solubility of the drug may explain the high concentrations found in ear wax and skin. Ivermectin was also detected in the body fluids and tissues of an untreated pig penned with the treated animals. Direct contact appeared to be necessary for transfer of ivermectin from the treated to the untreated pig but coprophagia or urine drinking is a possible explanation. The pharmacokinetics of ivermectin administered subcutaneously at a dose rate of 300 micrograms/kg to six pigs were studied. There was marked individual variation in the pharmacokinetics of ivermectin. In one pig the area under the plasma concentration-time curve was particularly high. This may reflect individual variation in uptake and excretion of the drug. The mean elimination half-life of the drug was 35.2 h, suggesting that the drug is cleared slowly from pigs with drug detectable in plasma for 6-10 days. This persistence should allow a short period of protection before re-infection with parasites.

Ultra-high-field MRI real-time imaging of HSC engraftment of the bone marrow niche.

The bone marrow (BM) undergoes extensive remodeling following irradiation damage. A crucial part of restoring homeostasis following irradiation is the ability of hematopoietic stem cells (HSCs) to home to and engraft specialized niches within the BM through a remodeling BM vascular system. Here we show that a combination of ultra-high-field strength magnetic resonance imaging (17.6 T, MRI) coupled with fluorescent microscopy (FLM) serves as a powerful tool for the in vivo imaging of cell homing within the BM. Ultra-high-field MRI can achieve high-resolution three-dimensional (3D) images (28 × 28 × 60 µm(3)) of the BM in live mice, sufficient to resolve anatomical changes in BM microstructures attributed to radiation damage. Following intra-arterial infusion with dsRed-expressing BM cells, labeled with superparamagnetic iron oxides, both FLM and MRI could be used to follow initial homing and engraftment of donor HSC to a limited number of preferred sites within a few cell diameters of the calcified bone-the endosteal niche. Subsequent histology confirmed the fidelity and accuracy of MRI to create non-invasive, high-resolution 3D images of donor cell engraftment of the BM in living animals at the level of single-cell detection.