Re-evaluation of the low-dose dexamethasone suppression test in dogs.

OBJECTIVES: This study aims to re-evaluate the low-dose dexamethasone suppression test 8-hour cortisol cut-point for the diagnosis of hypercortisolism in dogs using a solid-phase, competitive chemiluminescent enzyme immunoassay. MATERIALS AND METHODS: Twenty-seven client-owned dogs with naturally occurring hypercortisolism and 30 healthy control dogs were prospectively recruited. Performance of the low-dose dexamethasone suppression test was assessed using sensitivity, specificity and a receiver operating characteristic curve compared to a clinical diagnosis of hypercortisolism including response to treatment. RESULTS: Twenty-four dogs were diagnosed with pituitary-dependent hypercortisolism and three with adrenal-dependent hypercortisolism. In 30 healthy control dogs, 8-hour post-dexamethasone cortisol concentrations ranged from 5.5 to 39 nmol/L. A receiver operating characteristic curve curve constructed from the 8-hour post-dexamethasone cortisol concentrations of hypercortisolism and control dogs demonstrated that the most discriminatory cut-point was more than 39 nmol/L with sensitivity of 85.2% (95% confidence interval, 67.5% to 94.1%) and specificity of 100% (95% confidence interval, 88.7% to 100.0%) and an area under the curve of 0.963. CLINICAL SIGNIFICANCE: The optimal cut-point of more than 36 nmol/L proposed by this study is similar to the currently accepted 8-hour cortisol concentration cut-point for diagnosing hypercortisolism when using a solid-phase, competitive chemiluminescent enzyme immunoassay.

TRAIL responses are enhanced by nuclear export inhibition in osteosarcoma.

Tumour necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising anti-tumour agent that induces apoptosis of malignant cells through activation of death receptors. Death receptor agonistic antibodies are in clinical trials as TRAIL-mimetics, however, along with TRAIL monotherapy, there is limited efficacy due to the rapid emergence of TRAIL resistance, or due to existing TRAIL-insensitive disease. TRAIL-sensitisers, which enhance TRAIL activity or overcome TRAIL resistance, may facilitate death receptor agonists as viable anti-tumour strategies. In this study we demonstrate that the nuclear export inhibitor Leptomycin B, is a potent in vitro TRAIL-sensitiser in osteosarcoma cell lines. Leptomycin B works synergistically with both TRAIL and death receptor 5 agonistic antibodies to induce apoptosis in TRAIL sensitive cell lines. Further, Leptomycin B sensitises TRAIL-insensitive cell lines to TRAIL and death receptor agonistic antibody mediated apoptosis. We also confirmed that aldehyde dehydrogenase (ALDH) positive cells are not resistant to the apoptotic effects of TRAIL and Leptomycin B, an important observation since ALDH positive cells can have enhanced tumorigenicity and are implicated in disease recurrence and metastasis. The nuclear export pathway in combination with death receptor agonists, is a potential therapeutic strategy in osteosarcoma and warrants further research on clinically relevant selective inhibitors of nuclear export.

18 F-sodium fluoride positron emission tomography of the racing Thoroughbred fetlock: Validation and comparison with other imaging modalities in nine horses.

BACKGROUND: Early and accurate detection of stress remodelling in racehorses is of utmost importance to prevent catastrophic injuries. Current imaging techniques have limitations in assessing early changes predisposing to catastrophic breakdowns. Positron emission tomography (PET) using 18 F-sodium fluoride (18 F-NaF) is a sensitive method for the detection of early bone turnover and may improve early recognition of subtle injuries. OBJECTIVES: To validate the clinical use of 18 F-NaF PET in Thoroughbred racehorses, to assess the value of PET in the detection of bone lesions and to compare PET results with findings of other advanced imaging modalities, clinical examination and pathology. STUDY DESIGN: Experimental exploratory study. METHODS: Twenty fetlocks from nine Thoroughbred racehorses were imaged using 18 F-NaF PET, computed tomography (CT) and scintigraphy. Five fetlocks were also imaged with magnetic resonance imaging and four fetlocks were also examined histologically. Imaging findings were independently reviewed by three board certified radiologists. Imaging, clinical and histopathological findings were correlated. RESULTS: PET imaging was well-tolerated by all horses. PET detected focal areas of 18 F-NaF uptake in instances where other imaging modalities did not identify abnormalities, in particular in the proximal sesamoid bones. Maximal standardised uptake values could be measured to quantify the activity of lesions. Areas of 18 F-NaF uptake corresponded to regions of increased vascularity and increased osteoblastic activity. MAIN LIMITATIONS: Limited number of cases. CONCLUSIONS: 18 F-NaF PET imaging of the Thoroughbred fetlock is feasible and compares favourably with other imaging modalities in detecting stress remodelling in Thoroughbred racehorses. PET appears to be a beneficial imaging modality when used for early detection of stress remodelling in an effort to prevent catastrophic musculoskeletal injuries in this population of horses.

18 F-sodium fluoride positron emission tomography of the equine distal limb: Exploratory study in three horses.

BACKGROUND: Positron emission tomography (PET) is a cross-sectional, functional imaging modality that has recently become available to the horse. The use of 18 F-sodium fluoride (18 F-NaF), a PET bone tracer, has not previously been reported in this species. OBJECTIVES: To assess the feasibility of 18 F-NaF PET in the equine distal limb and explore possible applications in the horse in comparison with other imaging modalities. STUDY DESIGN: Exploratory descriptive study involving three research horses. METHODS: Horses were placed under general anaesthesia prior to intravenous (i.v.) administration of 1.5 MBq/kg of 18 F-NaF. Positron emission tomography imaging of both front feet and fetlocks was performed using a portable scanner. Computed tomography (CT) of the distal limb was performed under a separate anaesthetic episode. Bone scintigraphy and magnetic resonance imaging (MRI) were subsequently performed under standing sedation. Images obtained from PET and other imaging modalities were independently assessed and the results correlated. RESULTS: Positron emission tomography images were obtained without complication. The radiation exposure rate was similar to equine bone scintigraphy. Positron emission tomography detected focal 18 F-NaF uptake in areas where other imaging modalities did not identify any abnormalities. This included sites of ligamentous attachment, subchondral compact bone plate and the flexor cortex of the navicular bone. 18 F-NaF uptake was identified in some, but not all, osseous fragments and areas of osseous formation, suggesting a distinction between active and inactive lesions. MAIN LIMITATIONS: A small number of horses were included and histopathology was not available. CONCLUSIONS: 18 F-NaF PET imaging of the equine distal limb provides useful additional information when compared with CT, MRI and scintigraphy and has the potential for both research and clinical applications in the horse. The Summary is available in Chinese - see Supporting information.

Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes.

OBJECTIVE: These studies investigated cytokine and chemokine receptor profiles in nucleus pulposus (NP) cells, and the effects of receptor stimulation on mRNA levels of extracellular matrix (ECM) components, degrading enzymes and cytokine and chemokine expression. METHOD: Immunohistochemistry (IHC) was performed to localise expression of CD4, CCR1, CXCR1 and CXCR2 in human NP tissue samples. Effects of cytokine and chemokine stimulation was performed to investigate effects related to ECM remodelling and modulation of cytokine and chemokine mRNA expression. RESULTS: IHC identified CD4, CCR1, CXCR1 and CXCR2 expression by NP cells. Differential expression profiles were observed for CD4 and CXCR2 in tissue samples from degenerate and infiltrated IVDs. In vitro stimulations of primary human NP cultures with IL-16, CCL2, CCL3, CCL7 or CXCL8 did not identify any modulatory effects on parameters associated with ECM remodelling or expression of other cytokines and chemokines. Conversely, IL-1 was seen to modulate ECM remodelling and expression of all other cytokines and chemokines investigated. CONCLUSION: This study demonstrates for the first time that NP cells express a number of cytokine and chemokine receptors and thus could respond in an autocrine or paracrine manner to cytokines and chemokines produced by NP cells, particularly during tissue degeneration. However, this study failed to demonstrate regulatory effects on ECM genes and degradative enzymes or other cytokines and chemokines for any target investigated, with the exception of IL-1. This suggests that IL-1 is a master regulator within the IVD and may exert regulatory potential over a plethora of other cytokines and chemokines.

Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.

An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies.

A variety of gene therapy strategies are under development for the treatment of sickle cell anemia and other hemoglobinopathies. A number of alternative vectors have been developed to transfer and express the beta-globin gene and other therapeutic molecules, but none has resulted in efficient transduction and stable long-term expression in primary hematopoietic cells. One reason for this problem is that most vectors are initially evaluated in immortalized cell lines which may not faithfully recapitulate the biology of primary erythroid cells. In order to provide a more relevant system for efficiently evaluating alternative vector constructs for beta-globin disorders, we have developed (1) a simple method for generating primary human red blood cell (RBC) precursors in liquid culture established with mononuclear cells obtained from normal donors as well as patients with Hb SC disease; (2) a high titer retroviral vector which can be easily modified to optimize gene transfer and transgene expression; and (3) methods for transducing the RBC precursors at high efficiency. The development of simple and efficient methods and reagents for generating and transducing primary human RBC precursors provides a facile and effective means for screening alternative gene therapy strategies. Gene Therapy (2000) 7, 215-223.

Paroxysmal nocturnal hemoglobinuria: molecular pathogenesis and molecular therapeutic approaches.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic stem cell disorder classified as an intravascular hemolytic anemia. Abnormal blood cells are deficient in glycosylphosphatidyl inositol (GPI)-anchored proteins. Deficiencies of GPI-anchored complement regulatory proteins, such as decay accelerating factor (DAF) and CD59, render red cells very sensitive to complement and result in complement-mediated hemolysis and hemoglobinuria. In the affected hematopoietic cells from patients with PNH, the first step in biosynthesis of the GPI anchor is defective. Three genes are involved in this reaction step and one of them, an X-linked gene termed PIG-A, is mutated in affected cells. Granulocytes and lymphocytes from the same patient have the same mutation, indicating that a somatic PIG-A mutation occurs in hematopoietic stem cells. The PIG-A gene is mutated in all patients with PNH reported to date. We review these recent advances in the understanding of the molecular pathogenesis of PNH. Furthermore, we present an hypothesis regarding the predominance of the PNH clone, caused by positive selection by hematopoietic suppressive cytokines, such as transforming growth factor (TGF)-beta. In addition, we discuss the possibility of cure for PNH through molecular therapeutic strategy using gene transfer techniques. (Key words: paroxysmal nocturnal hemoglobinuria, glycosylphosphatidylinositol-anchored proteins, PIG-A, clonal dominance, growth advantage, transforming growth factor-beta, gene therapy, molecular therapeutic approach).

Multiparameter-fluorescence activated cell sorting analysis of retroviral vector gene transfer into primitive umbilical cord blood cells.

Retroviral vector gene transfer strategies are currently being developed to treat a variety of hematopoietic disorders. To date, genetic modification of human pluripotent hematopoietic stem cells has been inefficient. In the present study we developed reagents and procedures for rapidly screening retroviral vector gene transfer conditions using a multiparameter fluorescence-activated cell sorting (FACS) assay. To identify transduced cells using FACS analysis, we developed a retroviral vector, termed MN, which stably expressed high levels of a truncated version of the low-affinity nerve growth factor receptor (LNGFR). In addition, procedures were developed for enriching CD34+ cells from cryopreserved umbilical cord blood. These cells were transduced with MN and evaluated using multiparameter FACS analysis for expression of CD34, CD38, and LNGFR. Stem cell maintenance was determined by measuring the CD34hi and CD34hiCD38lo/- cells remaining after ex vivo gene transfer. Gene transfer into these cells was measured by evaluating cells expressing high levels of LNGFR. Initial studies with this assay and with in vitro functional assays indicated that retroviral gene transfer following pre-incubation with a variety of cytokines in serum containing conditions resulted in 1) poor maintenance of hematopoietic stem cells and 2) gene transfer predominantly in relatively mature cells. When gene transfer in serum-free conditions was performed, some improvement was observed in the maintenance of cells retaining primitive immunophenotypes with no reduction in the gene transfer efficiency. The MN vector and multiparameter FACS analysis will be useful in efficiently screening ongoing efforts designed to improve stem cell gene transfer.

Quantitative DNA ploidy analysis of breast carcinoma: a study of the effects of joint photographer experts group (JPEG) compression on DNA ploidy images.

Telepathology usage in the past has typically been a qualitative procedure rather than a quantitative measurement. DNA ploidy using image analysis has been favorably compared to DNA ploidy analysis by flow cytometry in numerous publications. A step from DNA ploidy analysis using conventional image analysis to DNA ploidy analysis using stored images allows DNA ploidy analysis by image cytometry to become a powerful tool in telepathology. Remote DNA ploidy analysis using stored images has an impact on the field of pathology, as not every hospital or laboratory can afford to perform this type of specialized testing. However, images have large data files and require lengthy transmission times over communication systems to other computers. Joint Photographer Experts Group (JPEG) compression is a computer algorithm that allows the file size of an image to be reduced in order to decrease transmission times to another computer. A study was initiated to investigate the effects of JPEG compression on images of Feulgen stained breast tumor touch preps and the resulting DNA ploidy histograms.

Quantitative DNA analysis: a comparison of conventional DNA ploidy analysis and teleploidy.

JPEG compression can be used on images for DNA ploidy analysis if careful consideration is given to the level of compression used for file storage. The amount of JPEG compression possible may vary depending on the type of tissue analyzed, however, a compromise may be reached for all types of tissue. JPEG compression should not go over a level of 70 for DNA analysis as this would result in possibly erroneous IOD calculation and erroneous DNA ploidy analysis. Also, the resulting file quality is so poor that even visual analysis is not possible. With careful training of personnel in cell selection, remote DNA ploidy analysis would be an effective tool for standardization and quality control in the pathology laboratory. By using remote DNA ploidy analysis, it is possible for hospitals to consolidate their workload, and make DNA ploidy analysis by image cytometry a cost effective test in the laboratory. Proficiency testing would also become possible as all laboratories performing DNA ploidy analysis would receive the same fields of view for testing. DNA ploidy analysis by image cytometry using stored images could be a versatile tool for the telepathology community.

Utilization review of treatment for chemical dependence.

Utilization review of treatment for chemical dependence has become increasingly important as evidence accumulates that outpatient treatment can be effective in many cases. The authors discuss the basic questions that guide utilization review of treatment for chemical dependence, summarize research that has led to the increasing emphasis on outpatient and partial hospital treatment approaches, and review the American Psychiatric Association's guidelines for utilization review of chemical dependence treatment. Case examples illustrate issues in the treatment of adolescents, patients with dual diagnoses, and relapsing patients. The authors conclude that utilization review supports use of inpatient care only for patients who require intensive services, including those who are a danger to themselves or others, have serious physical or psychiatric disorders, or have not responded to an adequate trial of outpatient treatment.

A survey of clinical isolates of Enterobacteriaceae using a series of DNA probes for aminoglycoside resistance genes.

DNA probes specific for the aminoglycoside adenylyltransferase ANT(2"), the aminoglycoside phosphotransferases APH(3')-I and APH(3')-II, and the aminoglycoside acetyltransferases AAC(3)-I, AAC(3)-V and AAC(6')-I were used to screen 151 clinical isolates for the presence of these resistance determinants. The two most common resistance genes in the study isolates were those encoding ANT(2") and APH(3')-I. The phenotypic pattern of aminoglycoside resistance, as determined by a modified disk diffusion test correlated well with the genotypes of the organisms as established using DNA probes, although strains carrying the ANT(2") gene could express one of several phenotypes.

Linkage analysis in the spinal muscular atrophy type of facioscapulohumeral disease.

Facioscapulohumeral disease is probably a heterogeneous disorder. We have ascertained and sampled two multigeneration families with the neurogenic form of this disorder, considered to be a type of spinal muscular atrophy (FSHSMA). The two families have 36 affected members. Linkage studies with 10 expressed and seven DNA restriction fragment length polymorphism (RFLP) markers failed to show significant linkage (Zmax greater than or equal to 3.00). However, two areas of probable linkage were defined on chromosomes 1p and 4q with the markers MNS (Zmax = 1.47 at theta max = 0.10) and PGM1 (Zmax = 0.94 at theta max = 0.001) respectively. We are using additional RFLPs from these and other areas of the human genome to screen these families for linkage to FSHSMA.

Chemical dependence treatment review guidelines.

During the last decade, there has been an explosive increase in the development of chemical dependence (CD) inpatient/freestanding units (Yahr: J Stud Alcohol 49:233-239, 1988) to the point that fully one third of all mental health admissions reviewed involve chemical dependence (L. Goldstein, personal communication; APA, Office of Quality Assurance, unpublished data). This dramatic increase has raised serious concerns among clinicians about the quality and appropriateness of treatment programs and among third-party payors about the cost of care. Five major categories should be considered when reviewing CD cases: (1) patient characteristics, including age, gender, severity of illness, course, past treatments, and impact of an intervention; (2) level of care, including outpatient, day treatment, freestanding units, hospital, and detoxification units; (3) match of services appropriate to patient's needs; (4) treatment program, including level of staff involvement, program elements, and planning for patient transition; and (5) peer review, including reviewing with a peer, being a patient advocate, and being a consultant. If these elements in these five categories are considered, a competent review is accomplished.

Development of a DNA probe from the deoxyribonucleotide sequence of a 3-N-aminoglycoside acetyltransferase [AAC(3)-I] resistance gene.

The aacC1 gene encoding the 3-N-aminoglycoside acetyltransferase [AAC(3)-I] was cloned from enteric plasmid pJR88, and its deoxyribonucleotide sequence was determined. Significant nucleotide homology was noted in the region extending from the proposed -35 sequences through the first 59 base pairs of the aacC1 gene open reading frame (ORF) and the upstream flanking regions and ORFs of several other antibiotic resistance genes. Sequences were noted to be homologous with the 6'-N-aminoglycoside acetyltransferase [AAC(6')-I], 2''-O-aminoglycoside adenylyltransferase [AAD(2'')], and 3''-O-aminoglycoside adenylyltransferase [AAD(3'')] resistance genes; the OXA-1, OXA-2, and PSE-2 beta-lactamase genes; and several dihydrofolate reductase genes. Small regions of homology were noted in the 3'-flanking regions of these resistance genes as well. A DNA probe for the aacC1 gene was selected from the nucleotide sequence information and was tested against a series of genetically and enzymatically defined strains. The probe, which proved specific for the aacC1 gene, was then tested against a series of 58 gentamicin-susceptible and 219 gentamicin-resistant gram-negative bacilli isolated from patients at the Seattle Veterans Administration Medical Center. Only six clinical isolates were noted to carry the aacC1 gene. Each was resistant to gentamicin but susceptible to kanamycin, tobramycin, and amikacin. The presence of homologous regions of DNA at both the 3' and 5' ends of the aacC1 gene reinforces the importance of choosing probes from within the ORFs of genes and of avoiding flanking sequences. When the homology with other sequences extends into the ORF, as it does with the aacC1 gene, development of a specific probe may require determination of the nucleotide sequence.

Cloning and sequencing of a gene encoding an aminoglycoside 6'-N-acetyltransferase from an R factor of Citrobacter diversus.

The aacA1 gene, which encodes a 6'-N-acetyltransferase [AAC(6')-I] mediating resistance to kanamycin, tobramycin, and amikacin, was cloned from the Citrobacter diversus R plasmid pBWH100 into the Escherichia coli vector pBR322. The complete nucleotide sequence of the gene and flanking regions was determined. A protein of approximately 21 kilodaltons was identified when the chimeric plasmid encoding the aacA1 gene was introduced into E. coli maxicells. This value is consistent with the size predicted for a protein translated from the open reading frame of the gene.