Long-term expression of gamma-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma-globin gene fused to the ankyrin-1 promoter

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked gamma-globin gene in transgenic mice. We inserted the Ank/(A)gamma-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/(A)gamma-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/(A)gamma-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse alpha-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe beta-thalassemia if the level of expression can be further increased.     

Small bowel enterocyte apoptosis and proliferation are increased in the elderly

BACKGROUND: It is known that in the elderly the small bowel does not reveal structural and functional deteriorations in normal conditions, whereas the absorptive function is impaired in stress conditions. OBJECTIVE: The balance between enterocyte apoptosis and proliferation being responsible for the maintenance of tissue size, mucosal morphology and function in the gastrointestinal tract, the aim of our study was to evaluate the rates of enterocyte apoptosis and proliferation in the duodenal mucosa of aged human beings in comparison to adults. METHODS: For this purpose, the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end labelling (TUNEL) technique and immunohistochemistry for MIB-1 detection were applied on histological sections of endoscopic duodenal biopsy specimens obtained from 12 healthy elderly subjects (mean age 77.6 years; M/F 7/5) and 12 healthy adult subjects (mean age 37.7 years; M/F 8/4). Counts were performed, at a constant magnification (x200), by computer-aided analysis and the results expressed as median percentages of positive enterocytes. RESULTS: The results showed a significant increase in enterocyte apoptosis in the elderly (15.3 vs. 2.1% in the adults, p < 0.001) which was positively correlated (r(s) = 0.65, p < 0.05) with a significant increase in enterocyte proliferation (37.7 vs. 15.8% in the adults, p < 0.0001). CONCLUSIONS: These data suggest that the maintenance of mucosal architecture throughout the process of aging is due to either a hyperproliferative state or an exaggerated apoptosis with a consequent cellular immaturity, which may impair the absorptive function observed in stress conditions.     

Stimulation with automatic radiofrequency scanning in the long term treatment of tachyarrythmias]

A pacemaker is described which uses radiofrequency signals for synchronisation and pacing, and seeks automatically the phase of the cycle at which the tachycardia may be interrupted. The patient himself can activate the pacemaker. The programme of the external transmitter, which scans nearly all the R-R cycles of the tachycardia, may be pre-set to transmit one or two synchronised stimuli, with a progressive delay of 5 or 10 ms. If the tachycardia is interrupted the pacing stops automatically. The implantable unit of the device may be connected to a normal endocardial electrode, has no batteries and is of small size and weight. The method has been used with success in 4 patients with reciprocal junctional tachycardia (3 of these patients had the Wolff-Parkinson-White syndrome), and in one case of chronic recurrent ventricular tachycardia. All of them were refractory to conventional therapy.     

Primary Nasal Tip Surgery: A Conservative Approach

Background

Rhinoplasty and rhinoseptoplasty are very important and complex surgical procedures because the nose plays a pivotal aesthetic role in the face and an important functional role in breathing. Mild bulbous, plunging, undefined tips are very common, and tip refining and repositioning often are required surgical procedures.

Methods

For 97 selected patients, the authors performed their personal technique consisting of a transcartilaginous approach, incomplete vertical interruption, and retrograde undermining of the lower lateral cartilages to improve tip projection and definition. The five aspects analyzed were nasal tip symmetry, nostril symmetry, tip projection, tip definition, and appearance of the nasal tip only. Each parameter was assessed using pre- and postoperative quantification according to a visual analog scale. Postoperative evaluation was performed during a mean follow-up period of 1?year.

Results

The results at 1?year showed high rates of improvement in tip definition (mean, 7.9?±?2.4) and nasal tip only evaluation (mean, 6.5?±?3.1). The patients reported an improvement in nasal tip symmetry (mean, 4.2?±?3.2), nostril symmetry (mean, 4.5?±?4.1), and tip projection (mean, 5.8?±?2.9).

Conclusions

This simple, safe, and effective technique is proposed for mild bulbous, plunging, undefined, and hypoprojected tips.     

Effects of desipramine and alprazolam in the forced swim test in rats after long-lasting termination of chronic exposure to picrotoxin and pentylenetetrazol

Rats were treated for 5 weeks with three subconvulsant doses of picrotoxin (PTX) and pentylenetetrazol (PTZ) per week to induce a persistent reduction of the GABA_A receptor function which results in chemical kindling. Fifteen days after termination of this treatment schedule, the effect of desipramine (DMI) and alpraxolam (ALP) on immobility time in the forced swim test (FST) was evaluated. Chronic PTX and PTZ did not alter the immobility time. Acute PTX and PTZ reduced the immobility of rats chronically treated with vehicle but not of those exposed chronically to PTX and PTZ. Chronic PTX did not influence the anti-immobility effect of DMI, but blocked that of ALP. Chronic PTZ markedly potentiated the anti-immobility effect of DMI but blocked that of ALP. Concomitant administration of chlordiazepoxide prevented the effects of chronic PTX and PTZ. These findings suggest that a long-lasting reduction in GABA_A receptor function, unlike acute reduction, does not play an important role in the mobility of rats in the FST and in the anti-immobility effect of DMI while it blocks that of ALP.     

Serum lipids and lipoproteins in patients treated with antiepileptic drugs

The concentration levels of serum lipids and lipoproteins were measured in three groups of children and adolescents treated with antiepileptic drugs: carbamazepine (14 patients), phenobarbital (20 patients), and valproic acid (20 patients). Patients treated with these drugs revealed significant changes in lipids and lipoproteins, but when the authors reevaluated the three groups of children 1 year after the end of treatment, a complete return to normal of all parameters was observed. These data demonstrate that the changes induced by these drugs are transient and reversible.     

Cortical stimulation and reflex excitability of spinal cord neurones in man

Summary The H reflex technique was used to evaluate the influence exerted by cortical conditioning on the excitability of the alpha-motoneurone pool and on IA interneuronal activity (reciprocal inhibition). In ten subjects at absolute rest electrical and magnetic stimulation of the motor cortex was transcranially applied during flexor carpi radialis H reflex eliciting and in conditions of reciprocal inhibition induced by radial nerve stimulation. The time courses showed that at intensities below motor threshold, electrical brain conditioning induced an increase in the amplitude of the test reflex when the cortical shock was given 4 ms after the test H reflex. On the contrary, reciprocal inhibition was reduced by electrical cortical conditioning when the scalp stimulation was applied 2–3 ms after the test stimulus. Magnetic transcranial stimulation induced an increase of H reflex amplitude when the test shock was administered 5 and 2 ms prior to the scalp shock; it did not modify the degree of reciprocal inhibition. The experimental findings could be considered the electrophysiological manifestation of a differential cortico-spinal control on the pathway alpha-motoneurone/IA interneurone. Considerations on the delay allow the hypothesis of a further synapse between the cortico-spinal ending and the IA interneurone.Discrepancies with magnetic conditioning might be ascribed to a preferential transynaptic action of magnetic mode of neural activation.This work was supported by grants from the Consiglio Nazionale delle Ricerche and the Ministero dell'Università e delia Ricerca Scientifica e Tecnologica, Roma, Italy     

Reduced frequency of wheezing respiratory illness in infants with perinatal human immunodeficiency virus-type 1 infection: A model for immunologic and inflammatory mechanisms of airway obstruction?

A multivariate analysis using a logistic regression model evaluated odds ratio (OR) and 95% confidence limits (95% CL) of pediatrician-diagnosed wheezing respiratory illness in 75 infants with perinatal human immunodeficiency virus-type 1 (HIV-1) infection, 205 uninfected infants of HIV-1 infected mothers, and 1780 infants of HIV-1 uninfected mothers. Infants were prospectively followed-up for the first 2 years of life. Covariates were risk factors for wheezing respiratory illness (preterm delivery, low birth weight, maternal smoking, formula feeding, and neonatal respiratory disorders). Maternal use of illicit drugs in pregnancy, antiretroviral treatment in pregnancy, maternal HIV-1-related clinical condition at the time of delivery were also included in the models when infants of HIV-1 infected mothers were taken into account. Although the frequency of risk factors for wheezing respiratory illness was higher in infants of HIV-1 infected than in those of uninfected mothers, HIV-1 infection emerged as a protective factor [OR: 0.001 (95% CL: 0.0001–0.01); p < 0.001]. The frequency of risk factors was similarly high among infants of infected mothers, but OR was lower in HIV-1 infected than in uninfected infants of infected mothers (0.005; 95% CL: 0.0004–0.06; p < 0.001). Finally, OR was higher in uninfected infants of HIV-1 infected mothers (who evidenced a higher frequency of risk factors) than in infants of HIV-1 uninfected mothers (9.97; 95% CL: 4.87–20.40; p < 0.001). Understanding the reason why HIV-1 protects against wheezing respiratory illness could shed light on the immunologic and inflammatory mechanisms of airway obstruction.     

Effects of imipramine on raphe nuclei and prefrontal cortex extracellular serotonin levels in the rat

 The effect of acute administrations of three doses of imipramine (1, 5 and 10 mg/kg SC), a widely used tricyclic antidepressant, on extracellular levels of serotonin (5-HT) has been studied by intracerebral microdialysis in raphe nuclei and prefrontal cortex of conscious rats. Imipramine 1 mg/kg SC did not change extracellular 5-HT in either raphe nuclei and prefrontal cortex. However, with the dose of 5 mg/kg SC imipramine induced in raphe nuclei, a brief increase of extracellular 5-HT followed by a lowering (55–65% basal release) of the neurotransmitter. The same dose of imipramine decreased (60–70% of basal value) extracellular 5-HT in prefrontal cortex. Imipramine 10 mg/kg SC significantly increased 5-HT levels in both raphe nuclei (190 ± 20% above basal value) and prefrontal cortex (280 ± 15% above basal value). Pretreatment with (-)pindolol (5 mg/kg SC), a non-selective 5-HT_1A subtype receptor antagonist, 30 min before imipramine 5 mg/kg, modified the effect of the antidepressant: an increase, instead of a decrease, on prefrontal cortex dialysate 5-HT was observed. (-)Pindolol (10 mg/kg SC) increased extracellular 5-HT in both raphe nuclei (155 ± 20% above basal value) and prefrontal cortex (160 ± 8% above basal value). These data show that acute administration of imipramine modifies extracellular 5-HT at the level of the raphe nuclei and prefrontal cortex. 5-HT_1A autoreceptors in the raphe nuclei, which this study suggests to be tonically active, may be stimulated after systemic administration of high doses of imipramine. Received: 10 April 1997 / Final version: 30 June 1997