An emerging consensus on recommendations to facilitate clinical gene transfer.
Explore the source record for details and available documents.
Consensus↗
Biomedical subjects
David A Williams
Publications and source records attributed to David A Williams.
The FIP1L1-PDGFRA fusion gene cooperates with IL-5 to induce murine hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL)-like disease.
Dysregulated tyrosine kinase activity by the Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha (PDGFRA) (F/P) fusion gene has been identified as a cause of clonal hypereosinophilic syndrome (HES), called F/P-positive chronic eosinophilic leukemia (CEL) in humans. However, transplantation of F/P-transduced hematopoietic stem cells/progenitors (F/P(+) HSCs/Ps) into mice results in a chronic myelogenous leukemia-like disease, which does not resemble HES. Because a subgroup of patients with HES show T-cell-dependent interleukin-5 (IL-5) overexpression, we determined if expression of the F/P fusion gene in the presence of transgenic T-cell IL-5 overexpression in mice induces HES-like disease. Mice that received a transplant of CD2-IL-5-transgenic F/P(+) HSC/Ps (IL-5Tg-F/P) developed intense leukocytosis, strikingly high eosinophilia, and eosinophilic infiltration of nonhematopoietic as well as hematopoietic tissues, a phenotype resembling human HES. The disease phenotype was transferable to secondary transplant recipients of a high cell dose, suggesting involvement of a short-term repopulating stem cell or an early myeloid progenitor. Induction of significant eosinophilia was specific for F/P since expression of another fusion oncogene, p210-BCR/ABL, in the presence of IL-5 overexpression was characterized by a significantly lower eosinophilia than IL-5Tg-F/P recipients. These results suggest that F/P is not sufficient to induce a HES/CEL-like disease but requires a second event associated with IL-5 overexpression.
Animals↗
HOXB4 inhibits cell growth in a dose-dependent manner and sensitizes cells towards extrinsic cues.
Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.
Animals↗
Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research.
OBJECTIVE: Lentiviral vectors are increasingly used for preclinical models of gene therapy and other forms of experimental transgenesis. Due to the broad tropism and the ability for concentration by ultracentrifugation, most lentiviral vector preparations are produced using the vesicular stomatitis virus glycoprotein (VSV-g) protein as envelope. Recently, Hanawa and colleagues have demonstrated that the ecotropic envelope protein of murine leukemia viruses allows efficient pseudotyping of HIV-1-derived vector particles. However, this method has found little acceptance, despite potential advantages. MATERIALS AND METHODS: We produced lentiviral vectors pseudotyped with murine ecotropic envelope using a four-plasmid transient transfection system and evaluated their performance in murine fibroblasts and hematopoietic cells. RESULTS: Titers of lentiviral "ecotropic" supernatants were only slightly lower than those produced with VSV-g, could be concentrated by overnight centrifugation (13,000g), and efficiently transduced murine fibroblasts and hematopoietic cells but not human cells. Our Institutional Biosafety Committee agreed on the production and use of replication-defective lentiviral vectors pseudotyped with murine ecotropic envelope under biosafety level 1 (BL1) conditions with additional BL2 practices. We also obtained useful guidelines for the work with human infectious lentiviral vectors. CONCLUSIONS: For the researcher, "ecotropic" lentiviral vectors significantly improve the convenience of daily work, compared to the conditions required for lentiviral pseudotypes that are capable of infecting human cells. High efficiency and superior biosafety in combination with convenient handling will certainly boost the potential applicability of this important vector system.
Animals↗
The role of chemokine activation of Rac GTPases in hematopoietic stem cell marrow homing, retention, and peripheral mobilization.
Signaling downstream from the chemokine receptor CXCR4, the tyrosine kinase receptor c-kit and beta1-integrins has been shown to be crucial in the regulation of migration, homing, and engraftment of hematopoietic stem cells and progenitors. Each of these receptors signal through Rac-type Rho guanosine triphosphatases (GTPases). Rac GTPases play a major role in the organization of the actin cytoskeleton and also in the control of gene expression and the activation of proliferation and survival pathways. Here we review the specific roles of the members of the Rac subfamily of the Rho GTPase family in regulating the intracellular signaling of hematopoietic cells responsible for regulation of homing, marrow retention, and peripheral mobilization.
Animals↗
Comparison of clinical and evoked pain measures in fibromyalgia.
UNLABELLED: Evoked pain measures such as tender point count and dolorimetry are often used to determine tenderness in studies of fibromyalgia (FM). However, these measures frequently do not improve in clinical trials and are known to be influenced by factors other than pain such as distress and expectancy. The purpose of this investigation was to determine whether evoked pain paradigms that present pressure stimuli in a random fashion (eg, Multiple Random Staircase [MRS]) would track with clinical pain improvement in patients with FM better than traditional measures. Sixty-five subjects enrolled in a randomized clinical trial of acupuncture were observed longitudinally. Clinical pain was measured on a 101-point numerical rating scale (NRS) and the Short Form McGill Pain Questionnaire (SF-MPQ), whereas evoked pressure sensitivity was assessed via manual tender point count, dolorimetry, and MRS methods. Improvements in clinical pain and evoked pain were assessed irrespective of group assignment. Improvement was seen in clinical pain during the course of the trial as measured by both NRS (P = .032) and SF-MPQ (P = .001). The MRS was the only evoked pain measure to improve correspondingly with treatment (MRS, P = .001; tender point count and dolorimeter, P > .05). MRS change scores were correlated with changes in NRS pain ratings (P = .003); however, this association was not stronger than tender point or dolorimetry correlations with clinical pain improvement (P > .05). Pain sensitivity as assessed by random paradigms was associated with improvements in clinical FM pain. Sophisticated pain testing paradigms might be responsive to change in clinical trials. PERSPECTIVE: Trials in fibromyalgia often use both clinical and experimental methods of pain assessment; however, these two outcomes are often poorly correlated. We explore the relationship between changes in clinical and experimental pain within FM patients. Pressure pain testing that applies stimuli in a random order is associated with improvements in clinical pain, but this association was not stronger than other experimental techniques.
Acupuncture Analgesia↗
Rational design and applications of a Rac GTPase-specific small molecule inhibitor.
Rac GTPases are involved in the regulation of multiple cell functions and have been implicated in the pathology of certain human diseases. Dominant negative mutants of Rac have been the tool of choice in studying Rac function in cells. Given the difficulty of introducing high concentrations of the Rac mutants into primary cells and nonspecific effects of the mutants on Rho guanine nucleotide exchange factor (GEF) activities, it is desirable to develop small molecule inhibitors that could specifically inhibit Rac activities. Here we describe the rational design, characterization, and applications of a first-generation Rac-specific small molecule inhibitor. On the basis of the structure-function information of Rac interaction with GEFs, in a computer-based virtual screening we have identified NSC23766, a highly soluble and membrane permeable compound, as a specific inhibitor of a subset of GEF binding to Rac and, therefore, Rac activation by these GEFs. In fibroblast cells, NSC23766 inhibited Rac1 GTP-loading without affecting Cdc42 or RhoA activity and suppressed cell proliferation induced by a Rac GEF Tiam1. It has little effect on cell growth induced by a constitutively active Rac1 mutant. In addition, NSC23766 inhibited: (1) the anchorage-independent growth and invasion phenotypes of human prostate cancer PC-3 cells; (2) Rac activation and Rac-dependent aggregation of platelets stimulated by thrombin; and (3) Rac1 and Rac2 activities of hematopoietic stem/progenitor cells and induced their mobilization from mouse bone marrow to peripheral blood. Thus, NSC23766 is a lead small molecule inhibitor of Rac activity and could be useful for studying Rac-mediated cellular functions and for modulating pathological conditions in which Rac-deregulation may play a role.
Adenocarcinoma↗
Utility of cognitive behavioral therapy as a treatment for insomnia in patients with fibromyalgia.
Explore the source record for details and available documents.
Adult↗
Stem cell mobilization by beta2-agonists.
Explore the source record for details and available documents.
Adrenergic beta-Agonists↗
Children are not little adults: just ask their hematopoietic stem cells.
HSCs differ during ontogeny in some important parameters, including anatomic site of residence and cell cycling characteristics. In this issue of the JCI, Bowie et al. show that postnatal HSCs as well as fetal liver HSCs in mice are active in the cell cycle at much higher rates than that of adult HSCs; however, this increased frequency of cycling abruptly ceases 4 weeks after birth (see the related article beginning on page 2808). The cycling postnatal HSCs expressed high levels of CXC chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1 [SDF-1]), a chemokine previously implicated in stem cell trafficking to the marrow cavity and shown to be expressed by cells within the hematopoietic microenvironment. These cells also possessed an engraftment defect impeding reconstitution in irradiated recipient mice, which was reversible by pretransplant administration of antagonists of the CXCL12 receptor, CXCR4. Such agents are currently clinically available, suggesting that this approach could be used to improve stem cell transplantation and engraftment.
Adult↗
Structure-function based design of small molecule inhibitors targeting Rho family GTPases.
Rho GTPases of the Ras superfamily are involved in the regulation of multiple cell functions and have been implicated in the pathology of various human diseases including cancer. They are attractive drug targets in future targeted therapy. A wealth of structure-function information made available by high resolution structures and mutagenesis studies has laid out the foundation for the derivation of a mechanism-based targeting strategy. Here we describe the rational design and characterizations of a first generation Rac-specific small molecule inhibitor. Based on the structure-function information of Rac interaction with GEFs, in a computer based Virtual Screening we have identified NSC23766, a highly soluble and membrane permeable compound, as a specific inhibitor of a subset of GEF binding to Rac and therefore Rac activation. In fibroblast cells NSC23766 inhibited Rac1 GTP-loading without affecting Cdc42 or RhoA activity and suppressed the Rac-GEF, Tiam1, and oncogenic Ras induced cell growth and transformation. NSC23766 also potently inhibited the prostate PC-3 cancer cell proliferation and invasion induced by Rac hyperactivation. Intraperitoneal administration of NSC23766 to laboratory mice resulted in effective Rac GTPase suppression and hematopoietic stem cell mobilization from the bone marrow to the peripheral blood, similar to the effects of genetically targeted disruption of Rac GTPases in the animals. A co-crystal structure of NSC23766 bound to Rac1 provided further insight for future medicinal chemistry modification and improvement of this lead Rac-specific inhibitor. Thus, structure-function based rational design may represent a new avenue for generating lead small molecule inhibitors of Ras superfamily GTPases that are useful for modulating pathological conditions in which the small GTPase deregulation may play a role.
Animals↗
Measurement of urinary 11-dehydro-thromboxane B2 excretion in dogs with gastric dilatation-volvulus.
OBJECTIVE: To measure 11-dehydro-thromboxane B2 (11-dTXB2) in urine of healthy control dogs, dogs undergoing ovariohysterectomy, and dogs with gastric dilatation-volvulus (GDV) and assess the relationship between urinary 11-dTXB2 concentrations in dogs with GDV and postoperative outcomes. SAMPLE POPULATION: Urine samples from 15 nonsurgical control dogs, 12 surgical control dogs, and 32 dogs with GVD. PROCEDURE: Urine samples were obtained from healthy pet dogs (ie, nonsurgical control dogs), dogs undergoing ovariohysterectomy at anesthetic induction and 1 hour following surgery (ie, surgical control dogs), and dogs with GDV at hospital admission and 1 hour following surgical derotation of the stomach (ie, GDV dogs). Urinary 11-dTXB2 concentrations were determined with an ELISA and normalized to urinary creatinine (Cr) concentrations by calculation of the 11-dTXB2 -to-Cr ratio. Differences in median 11-dTXB2 -to-Cr ratios among dogs and before and after surgery were analyzed. RESULTS: Urinary 11-dTXB2-to-Cr ratios did not differ between nonsurgical control dogs and surgical control dogs before or after surgery. Urinary 11-dTXB2-to-Cr ratios were significantly higher in GDV dogs at the time of hospital admission and 1 hour after surgery, compared with those of nonsurgical control dogs. Postoperative urine samples from GDV dogs had significantly higher 11-dTXB2-to-Cr ratios than postoperative urine samples from surgical control dogs. Median urinary 11-dTXB2-to-Cr ratios increased significantly in GDV dogs that developed postoperative complications. CONCLUSIONS AND CLINICAL RELEVANCE: Urinary 11-dTXB2 concentration is increased in GDV dogs at the time of hospital admission and after surgical derotation of the stomach, compared with that of healthy dogs. An increased urinary 11-dTXB2-to-Cr ratio following surgery is associated with an increased incidence of postoperative complications in dogs with GDV.
Animals↗
Serum lipase activities and pancreatic lipase immunoreactivity concentrations in dogs with exocrine pancreatic insufficiency.
OBJECTIVE: To determine serum lipase activities and pancreatic lipase immunoreactivity (PLI) concentrations in dogs with exocrine pancreatic insufficiency (EPI). ANIMALS: 74 healthy dogs and 25 dogs with EPI. PROCEDURES: A diagnosis of EPI was made on the basis of clinical signs, low serum trypsin like immunoreactivity (TLI) concentration, and response to treatment with enzyme replacement. Median values for fasting serum lipase activity and serum PLI concentrations were compared between the 2 groups with a Mann-Whitney U test. RESULTS: Median fasting serum lipase activity was not significantly different between dogs with EPI (366.0 U/L) and healthy dogs (294.5 U/L), and only 1 dog with EPI had a serum lipase activity less than the lower limit of the reference range. Median serum PLI concentration was significantly lower in dogs with EPI (0.1 microg/L) than in healthy dogs (16.3 microg/L). All dogs with EPI had serum PLI concentrations less than the lower limit of the reference range. CONCLUSION AND CLINICAL RELEVANCE: Serum lipase activity is not limited to the exocrine pancreas in origin, whereas serum PLI is derived only from the exocrine pancreas. Unlike in serum TLI concentrations, there was a small degree of overlap in serum PLI concentrations between healthy dogs and dogs with EPI. Serum TLI concentration remains the test of choice for diagnosis of EPI.
Animals↗
Development of a fecal sample collection strategy for extraction and quantification of fecal immunoglobulin A in dogs.
OBJECTIVE: To develop a fecal sample collection strategy and quantification method for measurement of fecal IgA concentrations in dogs. SAMPLE POPULATION: Fecal samples from 23 healthy pet dogs of various breeds. PROCEDURES: Immunoglobulin A was extracted from fecal samples. An ELISA for the measurement of fecal IgA concentrations was established and analytically validated. Intraindividual variation of fecal IgA was determined by calculation of coefficients of variation. A sample collection strategy was developed on the basis of results of intraindividual variation of fecal IgA concentrations. A reference range for fecal IgA concentrations was determined. RESULTS: The method for extraction and quantification of fecal IgA was determined to be sufficiently sensitive, reproducible, accurate, and precise. On the basis of the intraindividual variability of our results, the determined fecal sample collection strategy required analysis of a total of 4 fecal samples/dog, with each fecal sample collected on 2 consecutive days with 28 days between sample collection periods (ie, days 1 and 2 followed by days 28 and 29). Reference range values for fecal IgA concentration were 0.22 to 3.24 mg/g of feces. CONCLUSIONS AND CLINICAL RELEVANCE: Methods of fecal IgA extraction and quantification used in our study allow for identification of dogs with consistently low fecal IgA concentrations. Use of these techniques will enable future investigations into possible associations between low fecal IgA concentrations and signs of gastrointestinal disease in dogs.
Animals↗
Matrix metalloproteinase-9 activity in the cerebrospinal fluid and serum of dogs with acute spinal cord trauma from intervertebral disk disease.
OBJECTIVE: To detect matrix metalloproteinase (MMP)-9 in serum and CSF and determine relationships between MMP activity and severity of disease, duration of clinical signs, and duration of hospitalization in dogs with acute intervertebral disk disease (IVDD). ANIMALS: 35 dogs with acute IVDD and 8 clinically normal control dogs. PROCEDURE: CSF and serum were collected from affected and control dogs. Zymography was used to detect MMP-9. RESULTS: Activity of MMP-9 in CSF was detected in 6 of 35 dogs with IVDD; activity was significantly more common in dogs with duration of signs < 24 hours. Paraplegic dogs were more likely to have MMP-9 activity in the CSF than non-paraplegic dogs. No significant difference in hospitalization time was detected in dogs with IVDD between those with and without activity of MMP-9 in the CSF. Serum MMP-9 was detected more frequently in dogs with IVDD than in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Data were consistent with results of experimental rodent spinal cord injury studies that indicate that MMP-9 is expressed early during secondary injury.
Animals↗
Evaluation of gastrointestinal permeability and mucosal absorptive capacity in dogs with chronic enteropathy.
OBJECTIVE: To assess intestinal mucosal function by measuring permeability and absorptive capacity in dogs with chronic enteropathy (CE) before and after treatment and to determine whether those variables were correlated with clinical disease activity or histologic scoring of intestinal biopsy specimens. ANIMALS: 29 dogs with CE. PROCEDURE: Dogs were designated as having dietresponsive CE or CE requiring glucorticoid treatment. Severity of clinical signs was assessed by calculating the canine inflammatory bowel disease activity index (CIBDAI). Histologic severity of intestinal infiltration was assessed before and after 4 weeks of treatment in the diet-responsive group and before and after 10 weeks of treatment in the glucocorticoid group. Gastrointestinal permeability and mucosal absorptive capacity were assessed by use of intragastric administration of a solution containing lactulose, rhamnose, xylose, 3-O-methylglucose, and sucrose. Urine was collected 6 hours after administration of the sugar solution to determine urinary lactulose-to-rhamnose (L:R), xylose-to-methylglucose (X:M), and sucrose-to-methylglucose (S:M) ratios. RESULTS: Median CIBDAI scores decreased significantly in both groups of dogs after treatment. However, the median histologic grade of intestinal biopsy specimens did not change with treatment in either group. There were no significant differences in L:R, X:M, or S:M ratios after treatment in either group and no significant correlations between L:R, X:M, or S:M ratios and CIBDAI or histologic scores. CONCLUSIONS AND CLINICAL RELEVANCE: Results of tests for intestinal permeability and mucosal absorptive capacity were not useful indicators of clinical disease activity as assessed by the CIBDAI or the sever ity of infiltration as indicated by histologic evaluation.
Animals↗
Comparison of various doses of carbon 13-labeled aminopyrine for a carbon 13-labeled aminopyrine demethylation blood test in healthy dogs.
OBJECTIVE: To determine an optimal dose of carbon 13 ((13)C)-labeled aminopyrine for use in a (13)C-aminopyrine demethylation blood test in healthy dogs. ANIMALS: 9 adult dogs. PROCEDURES: Food was withheld from each dog for 12 hours. A 2-mL baseline blood sample was obtained from each dog and placed into an evacuated tube containing sodium heparin. Carbon 13-labeled aminopyrine was administered IV at doses of 1, 2, 5, or 10 mg/kg. Additional blood samples (2 mL) were obtained and placed into evacuated tubes containing sodium heparin 30, 45, 60, and 75 minutes after (13)C-aminopyrine administration. Hydrochloric acid was used to extract CO(2) from blood samples. The extracted gas was analyzed by fractional mass spectrometry to determine the percentage dose of (13)C administered as (13)C-aminopyrine and recovered in extracted gas (PCD). RESULTS: Gross evidence of clinical adverse effects was not detected in any dog after administration of (13)C-aminopyrine. The mean coefficient of variation (CV) for PCD was significantly lower than the mean CV for the summation of PCD values up to a given sampling time (CUMPCD). Mean PCD values among the 4 doses for each sample time were not significantly different. Administration of (13)C-aminopyrine at a dose of 2 mg/kg resulted in the lowest interindividual variability. CONCLUSIONS AND CLINICAL RELEVANCE: The PCD is superior to CUMPCD for the quantification of aminopyrine demethylation. Administration of (13)C-(13)C-aminopyrine at a dose of 2 mg/kg is appropriate for use in the (13)C-aminopyrine demethylation blood test in healthy dogs.
Aminopyrine↗