A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Electric activity in the neocortex of freely moving young and aged rats

Electroencephalographic activity of the neocortex was evaluated in young (5-7 months) and aged (26-28 months) rats. All animals in the aged group showed behavioral impairment in a spatial task (water maze). A neocortical electroencephalogram was derived simultaneously from 16 different neocortical locations and was subjected to spectral analysis. The frequency of occurrence and duration of high-voltage spindles was determined in two sessions, each involving a total of 30 min alert immobility. Changes in spectral characteristics and high-voltage spindles in response to scopolamine administration were also evaluated. The power of high-frequency activity (8-20 Hz) was significantly reduced in the aged subjects. This was greatest in the temporo-occipital regions, while no significant changes were seen in the mediofrontal region. Scopolamine resulted in a large power increase in all frequency bands, but the increase in the higher-frequency range (8-20 Hz) was significantly less in the aged group. The incidence of high-voltage spindles was 6 times higher and their total duration was 9 times longer in aged rats, with virtually no overlap with the young group. In young rats, scopolamine increased the incidence and total duration of high-voltage spindles, while it decreased both parameters in the aged subjects. Cholinergic neurons in the nucleus basalis appeared shrunken in the aged animals. These findings demonstrate that reliable electroencephalographic changes are present in the neocortex of the aged rat, and that some of the physiological alterations may be due to the pathological changes in the cholinergic nucleus basalis.     

α6 integrins participate in pro-T cell homing to the thymus

During embryogenesis, colonization of the thymic rudiment by hemopoietic progenitor cells depends on the adhesion of these cells to the jugular endothelium. Previously, we showed that progenitor T cells (pro-T cells) interact with α₆ integrins present on vascular endothelium. Here, we demonstrate that anti-α₆ integrin antibodies reduced the number of thymocytes up to 80 % in a congenic mouse model for thymus colonization by pro-T cells. In organotypic thymus cultures, the anti-α₆ integrin antibodies did not influence T cell development and proliferation. From this, we conclude that α₆ integrin participates in thymus homing. During mouse thymus ontogeny, α₆ integrin mRNA and protein expression was found as early as day 10 of development; at day 11, perithymic endothelial cells were α₆ integrin positive. Two α₆ integrin mRNA exist which are produced by alternative exon usage. The longer form, α₆, integrin, predominates during early embryonic stages, while the shorter α_6A form was present later during development. Although α₆, integrins can be displayed by immature thymocytes, strongest expression was found on intra- and perithymic vascular endothelium. These data suggest that α₆ integrins are involved in the homing of pro-T cells to the developing thymus by mediating adhesion of pro-T cells to the vascular endothelium.     

Apoptosis in breast carcinoma

Apoptosis may play a major role in determining tumor growth and aggressiveness. The aim of this study was to examine the relationship between apoptosis, expression of bcl-2 and p53 proteins, proliferation index, and other clinicopathological features of breast carcinoma. Sixty-five formalin-fixed paraffin-embedded tissue sections from invasive ductal breast carcinomas were studied for the presence of apoptosis by the terminaldeoxynucleotidyl-transferase-mediated dUTP-FITC nick end-labeling (TUNEL) method. Immunohistochemical methods were also used to determine the expression of estrogen receptor, Ki67, bcl-2 and p53 proteins. The number of apoptotic cells ranged from 2.0 to 236.0/10HPF (mean 36.26, median 28.0). The observation of 30 apoptotic cells/10HPF was more common in tumors > 3 cm, of histological grade III, with a high mitotic index, Ki67 index > or = 300, and p53 positivity; however, statistical significance was found only for the histological grade. Grade I and III tumors displayed an inverse association between the apoptotic index and bcl-2 and p53 protein expressions; grade I tumors frequently expressed bcl-2 (19/28), lacked p53 (20/28), and presented a low number of apoptotic cells (18/28), whereas grade III tumors tended to express p53 (12/17), lacked bcl-2 (13/17), and displayed a high number of apoptotic cells/10HPF (12/17). Multivariate analysis for survival revealed that estrogen receptors and apoptosis were independent variables. These data suggest that apoptosis, rather than proliferation index or expression of bcl-2 or p53 proteins, is an independent factor for the prognosis of survival.     

The orientation of horizontal cell dendrites in the superior colliculus of the hamster: an analysis based on three-dimensional reconstruction of intracellularly injected neurons

Summary We have employed intracellular injection of horseradish peroxidase (HRP) and 3-dimensional, computer-assisted reconstruction to delineate the organization of the dendrites of horizontal cells in the superficial laminae (the stratum griseum superficiale-SGS, and stratum opticum-SO) of the hamster's superior colliculus. Fifteen well-filled cells were analyzed. The dendrites of these cells were generally parallel to the frontal plane. An average of 74.8±13.0% of the total dendritic arbor of the recovered horizontal cells was located within 30° of this plane. The long axis of horizontal cell dendritic trees deviated an average (mean ± s.d.) of 21.7±13.2° from the frontal plane and the average extent of the dendritic tree in this plane was 637±216 m. This differed significantly from the average dendritic extent in the rostrocaudal axis (358±146 m, p<0.001). In some cases, portions of the dendritic arbors of horizontal cells appeared to be oriented along lines of isoelevation or isoazimuth of the visual field representation in the superficial laminae. For other cells, there was no clear relationship between dendritic orientation and the visual field map.     

Silent ovulation in rhesus monkeys (M. mulatta): dissociation of hormonal and behavioral states

Ovarian hormone levels were monitored in rhesus monkeys during the breeding and non-breeding seasons. Ovulation (as inferred from progesterone levels in blood serum in excess of 1.5 ng/ml) was most frequent in the breeding season but was absent only during the month of August. Summer (non-breeding season) ovulations were more frequently "silent," or without heterosexual behavior, than were winter ovulations. This finding suggested that estradiol stimulation may cause different behavioral responses at different times of the year. To test this hypothesis, ovariectomized females were treated with subcutaneously implanted estradiol capsules at different times within and outside the normal breeding season. In either season, treatment with estradiol resulted in increased female-to-female sexual behavior. Only during the breeding season, however, did the estradiol-treated ovariectomized females interact sexually with males. Although males copulated with both estradiol-treated and gonadally intact, untreated females during the breeding season, their rate of copulation with the intact females was higher. The results suggest that: (1) Some female rhesus monkeys displaying strictly seasonal breeding behavior continue to have luteal activity indicative of ovulation in May, June and July, (2) Summer ovulatory cycles are rarely accompanied by sexual behavior, (3) Estradiol treatment of ovariectomized females induces female-to-female sexual behavior both in the breeding and non-breeding seasons, and (4) Males are less responsive to the presence of estrogen-stimulated females in the non-breeding season than in the breeding season.     

Preoperative management of obese patients undergoing bariatric surgery: Role of endoscopy and Helicobacter eradication

BackgroundObesity is a pandemic disease associated to severe health problems. Management is usually multimodal, but many patients eventually need surgery to reduce weight. Many guidelines recommend endoscopy prior to surgery. This study reviews a series of patients undergoing sleeve gastrectomy to see whether endoscopy performance and histopathological findings influence surgery outcome.Material and methodsRetrospective series of patients undergoing sleeve gastrectomy as bariatric procedure at a single institution. We have reviewed the demographic data, the associated pathologies, endoscopic findings prior to surgery, histopathological findings in the surgical resection specimen and postoperative complication rate.Results259 patients fulfilled criteria for the study. Over 70% were women and the mean age was 46.9 (SD 9.8). Preoperative endoscopy was performed in 28.9% of the patients and biopsy only in 19.3%. Helicobacter pylori was detected in 28% of the patients undergoing endoscopy (either in the biopsy or the urease test) and eradicated before surgery in all the patients. Helicobacter pylori was present in 9.7% of the surgical resection specimens and its presence was significantly associated with the development of postoperative complications, mostly staple line leaks (p = 0.01).ConclusionOur study confirms that Helicobacter infection is significantly associated with postoperative complications after sleeve gastrectomy. It is therefore important to detect its presence and eradicate it before surgery.     

Body Composition Changes after a Weight Loss Intervention: A 3-Year Follow-Up Study

Studies comparing different types of exercise-based interventions have not shown a consistent effect of training on long-term weight maintenance. The aim of this study was to compare the effects of exercise modalities combined with diet intervention on body composition immediately after intervention and at 3 years’ follow-up in overweight and obese adults. Two-hundred thirty-nine people (107 men) participated in a 6-month diet and exercise-based intervention, split into four randomly assigned groups: strength group (S), endurance group (E), combined strength and endurance group (SE), and control group (C). The body composition measurements took place on the first week before the start of training and after 22 weeks of training. In addition, a third measurement took place 3 years after the intervention period. A significant interaction effect (group × time) (p = 0.017) was observed for the fat mass percentage. It significantly decreased by 5.48 ± 0.65%, 5.30 ± 0.65%, 7.04 ± 0.72%, and 4.86 ± 0.65% at post-intervention for S, E, SE, and C, respectively. Three years after the intervention, the fat mass percentage returned to values similar to the baseline, except for the combined strength and endurance group, where it remained lower than the value at pre-intervention (p < 0.05). However, no significant interaction was discovered for the rest of the studied outcomes, neither at post-intervention nor 3 years later. The combined strength and endurance group was the only group that achieved lower levels of fat mass (%) at both post-intervention and 3 years after intervention, in comparison with the other groups.