Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline

The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology.     

Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into ‘neighborhoods’ comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.     

Neurobehavioral Symptoms and Family Functioning in Traumatically Brain-Injured Adults

Traumatic brain injury (TBI) often results in a myriad of symptoms across physical, cognitive, and neurobehavioral domains. Despite inherent limitations associated with physical or cognitive impairments, the extant literature suggests that neurobehavioral symptoms tend to be the most distressing symptoms for the family and are more strongly related to poor outcome for the patient. The Neuropsychology Behavior and Affect Profile (NBAP) along with the General Functioning subscale of the Family Assessment Device (FAD-GF) and the Perceived Stress Scale were administered to 153 family members of persons who had sustained a TBI. The results provide new normative data and statistical support for the NBAP as a promising measure of neurobehavioral symptomatology following TBI. The correlation of.54 (p <.01) between FAD-GF and Full Scale NBAP scores provides powerful support for the hypothesis that family dysfunction is related to the presence of neurobehavioral symptoms in the patient. NBAP domains of Depression, Inappropriateness, Pragnosia, and Indifference appear most strongly related to family functioning and also bear a significant relationship to caregiver stress level and patient unemployment, whereas injury severity had little impact on either family functioning or neurobehavioral symptoms. The findings reinforce the significance of neurobehavioral symptoms and fortify their proposed link to family dysfunction post-TBI.     

Fuel kinetics during intense running and cycling when fed carbohydrate

On two occasions, six well-trained, male competitive triathletes performed, in random order, two experimental trials consisting of either a timed ride to exhaustion on a cycle ergometer or a run to exhaustion on a motor-driven treadmill at 80% of their respective peak cycling and peak running oxygen (VO_2max) uptakes. At the start of exercise, subjects drank 250 ml of a 15 g·100 ml^–1 w/v [U-¹⁴C]glucose solution and, thereafter, 150 ml of the same solution every 15 min. Despite identical metabolic rates [VO₂ 3.51 (0.06) vs 3.51 (0.10) 1·min^–1; values are mean (SEM) for the cycling and running trials, respectively], exercise times to exhaustion were significantly longer during cycling than running [96 (14) vs 63 (11) min; P < 0.05]. The superior cycling than running endurance was not associated with any differences in either the rate of blood glucose oxidation [3.8 (0.1) vs 3.9 (0.4) mmol· min^–1], or the rate of ingested glucose oxidation [2.0 (0.1) vs 1.7 (0.2) mmol· min^–1] at the last common time point (40 min) before exhaustion, despite higher blood glucose concentrations at exhaustion during running than cycling [7.0 (0.9) vs 5.8 (0.5) mmol·1^–1; P < 0.05]. However, the final rate of total carbohydrate (CHO) oxidation was significantly greater during cycling than running [24.0 (0.8) vs 21.7 (1.4) mmol C₆·min^–1; P < 0.01]. At exhaustion, the estimated contribution to energy production from muscle glycogen had declined to similar extents in both cycling and running [68 (3) vs 65 (5)%]. These differences between the rates of total CHO oxidation and blood glucose oxidation suggest that the direct and/or indirect (via lactate) oxidation of muscle glycogen was greater in cycling than running.     

Detection of Leishmania infantum kinetoplast DNA in laryngeal tissue from an immunocompetent patient

Mucosal leishmaniasis of the upper respiratory tract is usually associated with the visceral form or is found in immunosuppressed individuals. This report presents a case of isolated mucosal leishmaniasis in an immunocompetent patient, whose diagnosis mainly rested on histology and positive polymerase chain reaction result for Leishmania donovani in the laryngeal tissue. A 59-year-old man, who never lived outside Italy, showed a subglottic mucosal polypoid-like lesion. The typical morphological picture and positive polymerase chain reaction result for L donovani by DNA extracted from laryngeal biopsy specimens allowed the diagnosis of mucosal leishmaniasis. Specific amphotericin B therapy was started, resulting in clinical and endoscopic improvement. Increased knowledge about the histological and molecular tissue analysis of Leishmania enhances the diagnostic testing for mucosal leishmaniasis, as primary mucosal leishmaniasis may occur in both immunosuppresed and immunocompetent patients who travel to or reside in areas endemic for Leishmania.     

An analytical model for the dissolution of different particle size samples of Bioglass in TRIS-buffered solution

We analyzed the early stages of reactivity of three different particle size samples of Bioglass 45S5 and a bulk sample in TRIS-buffered solution at pH 8. Ion release, measured with ion-coupled plasma emission spectroscopy, and pH variations are reported. It was demonstrated that differences in the initial surface area influence the increase in pH, the rate of elemental release, and the rate of calcium phosphate reprecipitation. In particular, a thicker Ca/P layer was obtained on larger particles. The equilibrium value of Si in solution was independent of sample form and amount of sample dissolved, and was always close to the value observed when bulk silica is dissolved at pH 8. An analytical model is proposed for cation release, based on a two-step mechanism. It was found that the early stage of dissolution was nearly diffusion controlled for larger particles and bulk samples. The second stage was similar to a first-order homogeneous dissolution. The influence of sample surface area/solution volume ratio seemed to be more complex than that proposed in the early works presented in the literature. It is suggested that variation of surface area has a significant impact on the course of the dissolution.     

CYFIP2 is highly abundant in CD4+ cells from multiple sclerosis patients and is involved in T cell adhesion

DNA microarray profiling of CD4(+) and CD8(+) cells from non-treated relapsing and remitting multiple sclerosis (MS) patients determined that the cytoplasmic binding partner of fragile X protein (CYFIP2, also called PIR121) was increased significantly compared to healthy controls. Western analysis confirmed that CYFIP2 protein was increased approximately fourfold in CD4(+) cells from MS compared to inflammatory bowel disorder (IBD) patients or healthy controls. Because CYFIP2 acts as part of a tetrameric complex that regulates WAVE1 activation we hypothesized that high levels of CYFIP2 facilitate T cell adhesion, which is elevated in MS patients. Several findings indicated that increased levels of CYFIP2 facilitated adhesion. First, adenoviral-mediated overexpression of CYFIP2 in Jurkat cells increased fibronectin-mediated adhesion. Secondly, CYFIP2 knock-down experiments using antisense oligodeoxynucleotides reduced fibronectin-mediated binding in Jurkat and CD4(+) cells. Thirdly, inhibition of Rac-1, a physical partner with CYFIP2 and regulator of WAVE1 activity, reduced fibronectin-mediated adhesion in Jurkat and CD4(+) cells. Finally, inhibition of Rac-1 or reduction of CYFIP2 protein decreased fibronectin-mediated adhesion in CD4(+) cells from MS patients to levels similar to controls. These studies suggest that overabundance of CYFIP2 protein facilitates increased adhesion properties of T cells from MS patients.     

Effects of nitric oxide synthase inhibition by l-NAME on oxygen uptake kinetics in isolated canine muscle in situ

We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups ( n = 8 and 6) of healthy young men were studied over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload ( W _max). To label tissue proteins in muscle and tendon primed, constant infusions of [1-¹³C]leucine or [1-¹³C]valine and flooding doses of [¹⁵N] or [¹³C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography–mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h⁻¹), muscle collagen (0.054% h⁻¹), myofibrillar protein (0.121% h⁻¹), and sarcoplasmic protein (0.134% h⁻¹)). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation.     

Clinical impact of a 2-week psychotropic medication washout in unipolar depressed inpatients

BACKGROUND: Short-term discontinuation of psychiatric medications is required in many types of research studies. Yet there are few studies of the clinical impact of psychotropic discontinuation. We studied the impact of a short-term medication washout in unipolar depressed patients consecutively admitted to hospital for neuroimaging and cerebrospinal fluid (CSF) studies. METHOD: Patients (n=51) with unipolar depression who were taking antidepressant or mood stabilizing medication at or within 1 week of admission, and who had not been responding satisfactorily, were assessed for severity of psychopathology within 1 week of hospital admission and 41 of the group were reassessed following an approximately 2-week medication washout. RESULTS: On average, patients remained stable during the washout or improved on clinical measures. No serious adverse clinical changes were observed. LIMITATIONS: Potential sample bias, small sample size. CONCLUSION: The results suggest that similar studies can be conducted without causing undue worsening of symptoms. The benefit of medication washout may be related to the fact that many of the patients had been responding poorly to the medication they were taking. There is a need for further research on the effects of medication washout, for example in outpatients or those who are responding well to treatment, but have intolerable side-effects.