Genomic rearrangements at the <Emphasis Type="Italic">IGHMBP2</Emphasis> gene locus in two patients with SMARD1

Autosomal recessive spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by mutations in the immunoglobulin -binding protein 2 (IGHMBP2) gene. Patients affected by the infantile form of SMARD1 present with early onset respiratory distress. So far, patients with neither juvenile onset nor with larger deletions/rearrangements in IGHMBP2 have been reported. In this study, we investigated one patient with infantile (4 months) and another with juvenile (4.3 years) onset of respiratory distress. Direct sequencing of all exons and flanking intron sequences in both patients revealed a mutation on only one allele. In both patients, we identified genomic rearrangements of the other allele of IGHMBP2 by means of Southern blotting. Putative breakpoints were confirmed by polymerase chain reaction on genomic and cDNA. The patient with juvenile onset had an Alu/Alu mediated rearrangement, which resulted in the loss of ~18.5 kb genomic DNA. At the mRNA level, this caused an in-frame deletion of exons 3–7. The patient with infantile onset had a complex rearrangement with two deletions and an inversion between intron 10 and 14. This rearrangement led to a frameshift at the mRNA level. Our results show that SMARD1 can be caused by genomic rearrangements at the IGHMBP2 gene locus. This may be missed by mere sequence analysis. Additionally, we demonstrate that juvenile onset SMARD1 may also be caused by mutations of IGHMBP2. The complex nature of the genomic rearrangement in the patient with infantile SMARD1 is discussed and a deletion mechanism is proposed.     

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein–based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia

Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4⁺ (1/10) and CD8⁺ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8⁺ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4⁺ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.     

The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

Sleep patterns of day-working, evening high-schooled adolescents of São Paulo, Brazil

Children who grow up in developing countries of the world must work to help financially support their families, and they must also attend school. We investigated the impact of work on the sleep of working vs. nonworking high school students. Twenty-seven S?o Paulo, Brazil, public high school students (eight male and eight female working students plus six nonworking female and five nonworking male students) 14-18 yrs of age who attended school Monday-Friday between 19:00 to 22:30h participated. A comprehensive questionnaire about work and living conditions, health status, and diseases and their symptoms was also answered. The activity level and rest pattern (sleep at night and napping during the day) were continuously assessed by wrist actigraphy (Ambulatory Monitoring, USA). The main variables were analyzed by a two-factor ANOVA with application of the Tukey HSD test for multiple comparisons, and the length of sleep during weekdays vs. weekends was compared by Student t-test. Working students went to sleep earlier weekends [F(1,23)=6.1; p=0.02] and woke up earlier work days than nonworking students [F(1,23) = 17.3; p = 0.001]. The length of nighttime sleep during weekdays was shorter among all the working [F(1,23)= 16.7; p <0.001] than all the nonworking students. The sleep duration of boys was shorter than of girls during weekends [F(1,23)= 10.8; p <0.001]. During weekdays, the duration of napping by working and nonworking male students was shorter than nonworking female students. During weekdays working girls took the shortest naps [F(1,23)= 5.6; p = 0.03]. The most commonly reported sleep complaint during weekdays was difficulty waking up in the morning [F(1,23) = 6.5; p = 0.02]. During weekdays, the self-perceived sleep quality of working students was worse than nonworking students [F(1,23) = 6.2; p = 0.02]. The findings of this study show that work has negative effects on the sleep of adolescents, with the possible build-up of a chronic sleep debt with potential consequent impact on quality of life and school learning.     

Mechanotransduction of hemodynamic forces regulates organogenesis

Endothelial cells (EC) of the vertebrate cardiovascular system (CVS) are bona fide, yet enigmatic mechanoreceptors. When cultured in vitro and exposed to fluid forces, EC modify their physiological behaviour at the structural, metabolical and gene expression levels in response to the mechanical stimulus. However, as a direct consequence of the hypoxic bias (and often the lethality) that results from blocking blood flow in most animal systems, the in vivo role of EC mechanosensation (ECMS) remains poorly understood. The zebrafish has recently emerged as an alternative genetic model for the study of vertebrate development. Its striking ability to survive until larval stages in the absence of blood circulation circumveys the usual caveats that are inherent to CVS research, and offers the exciting opportunity to dissect the function of ECMS in vivo. Two groups have already uncovered an essential role for ECMS in zebrafish organogenesis, particularly in heart morphogenesis. In embryos in which intracardiac blood flow is genetically or physically compromised, several features of the normally developing heart, including valve formation, are specifically disrupted. In addition, impressive imaging studies of zebrafish hemodynamics demonstrate that the shear stress exerted upon the cardiac endothelium is largely in the range of the stimulus that in vitro activates cytoskelettal remodeling and gene expression changes in EC. Hence the cardiac phenotypes observed in vivo may indeed directly result from a lack of ECMS-dependent EC activity. These data shed first light on the role of ECMS in vivo. Notably, they also suggest that a number of human congenital cardiomyopathies may arise through abnormal fetal hemodynamics and/or EC sensory activity. Finally, these discoveries reinforce the too often neglected role of epigenetic factors (in this case, fluid forces) in the regulation of animal development.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Liposome-stabilized oil-in-water emulsions as adjuvants: increased emulsion stability promotes induction of cytotoxic T lymphocytes against an HIV envelope antigen

Protective or therapeutic immunity against HIV infection is currently believed to require both antibody and CTL responses against the envelope protein. In the present study, the adjuvant activity of a unique oil-in-water emulsion, in which liposomes containing lipid A (LA) and encapsulated antigen served as the emulsifying agent, was examined in mice using oligomeric gp140 (ogp140) derived from the HIV-1 envelope as the antigen. Emulsions rendered either highly stable or unstable by altering the ratio of liposomes to oil were used to examine the effect of stability of the emulsion on adjuvant activity. Stable and unstable emulsions had similar potencies for inducing both IgG antibodies to ogp140 and antigen-specific T-lymphocyte proliferation. Stable emulsions, but not unstable emulsions, induced antigen-specific CTL responses, possibly because of the depot effect of the stable emulsions. Furthermore, stable emulsions induced lower IgG2a/IgG1 ratios than the unstable emulsions. We conclude that stable liposomal oil-in-water emulsions provide an effective means of obtaining both antibody and CTL responses against an HIV envelope antigen.     

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.