From Lowe syndrome to Dent disease: correlations between mutations of the OCRL1 gene and clinical and biochemical phenotypes

Mutations of OCRL1 are associated with both the Lowe oculocerebrorenal syndrome, a multisystemic and Dent-2 disease, a renal tubulopathy. We have identified a mutation in 130 Lowe syndrome families and 6 affected by Dent-2 disease with 51 of these mutations being novel. No founding effect was evidenced for recurrent mutations. Two mutations initially reported as causing Dent-2 disease were identified in patients, including two brothers, presenting with Lowe syndrome thus extending the clinical variability of OCRL1 mutations. mRNA levels, protein content, and PiP(2) -ase activities were analyzed in patient's fibroblasts. Although mRNA levels were normal in cells harboring a missense mutation, the OCRL1 content was markedly lowered, suggesting that enzymatic deficiency resulted mainly from protein degradation rather than from a catalytic inactivation. Analysis of a splicing mutation that led to the elimination of the initiation codon evidenced the presence of shortened forms of OCRL1 that might result from the use of alternative initiation codons. The specific mapping of the frameshift and nonsense mutations, exclusively identified in exons 1-7 and exons 8-23, respectively, for Dent disease and Lowe syndrome together with the possible use of alternative initiation codons might be related to their clinical expression, that is, Lowe syndrome or Dent-2 disease.     

Effects of serum-free culture at the air-liquid interface in a human tissue-engineered skin substitute

Previous studies have reported that well-defined culture conditions can improve keratinocytes terminal differentiation and reproducibility. The aim of our study was to compare skin substitutes cultured in a complete medium with those cultured in a serum-free medium at the air-liquid interface to optimize the self-assembly method. Skin substitutes, cultured in a serum-free medium over 7, 14, and 21 days, were compared with others cultured in a complete medium (5% serum) over the complete culture period. Masson's Trichrome staining showed that the substitutes cultured in a serum-free medium generated a well-developed and differentiated epidermis. Immunolabeling analyses between the substitutes cultured without serum and those cultured in complete serum showed similar expression of epidermal differentiation markers, dermo-epidermal junction, and dermal extracellular matrix components. On the basis of our Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) results, the skin substitutes cultured in serum-free condition over 21 days of culture at the air-liquid interface showed lower frequencies of the CH(2) symmetric mode of vibrations, which means a better lipid organization of the stratum corneum. No significant difference in hydrocortisone penetration was observed between serum-free medium substitutes and the controls. Results demonstrate that the absence of serum does not compromise the characteristics of the skin substitutes observed in this study.     

Genetic sequence variations and <Emphasis Type="Italic">ADPRT</Emphasis> haplotype analysis in French Canadian families with high risk of breast cancer

The poly(ADP-ribose) polymerase (PARP/ADPRT) protein family catalyzes the synthesis of cellular poly(ADP-ribose) following DNA damage and is involved in genomic integrity by regulating cellular responses to DNA damage and apoptosis. Moreover, ADPRT inhibition contributes to a protective effect against cancer development. These findings render ADPRT an attractive candidate susceptibility gene for breast cancer, and thus the goal of this study was to evaluate the possible involvement of ADPRT sequence variations in breast cancer susceptibility. The complete sequence of the 23 exons and flanking intronic sequences of the ADPRT gene was analyzed in 54 affected individuals from distinct high-risk non-BRCA1/2 French Canadian families. No deleterious truncating mutation was identified in the coding region. However, 34 sequence variations were identified, among which seven are coding variants and seven are novel changes. All coding variants and intronic changes located in the vicinity of the coding variants identified in the case series were also analyzed in a cohort of 73 unrelated healthy French Canadian individuals. Interestingly, one missense variant (Pro377Ser) was observed in three different breast cancer cases but was not present among unaffected individuals. We have conducted here an exhaustive detailed mutation and haplotype tagging analysis of the ADPRT gene with regard to breast cancer, providing useful data for other large-scale association studies. Additional studies in other cohorts and other populations are however needed to further evaluate the implication of the Pro377Ser missense variant with regard to breast cancer susceptibility. Other members of INHERIT BRCAs involved in clinical aspects of the study are listed in the Appendix. J. Simard holds a Canada Research Chair in Oncogenetics.     

Use of short-acting beta2-agonists during pregnancy and the risk of pregnancy-induced hypertension

BACKGROUND: The effect of inhaled short-acting beta(2)-agonists (SABAs) on pregnancy outcome, especially hypertensive complications, is not well documented. After the finding of a possible protective association of inhaled SABAs with pregnancy-induced hypertension (PIH) in a previous study, we decided to further investigate their effect on this condition. OBJECTIVE: We sought to determine the effect of inhaled SABA use during pregnancy on the risk of PIH (gestational hypertension, preeclampsia, or eclampsia) in asthmatic women. METHODS: Three of Quebec's administrative databases were linked to constitute a cohort of asthmatic women who had at least 1 delivery between 1990 and 2000. A nested case-control study was performed using up to 10 control subjects matched to each case patient for the year of conception and gestational age. Statistical analyses considered 22 confounders. RESULTS: The cohort was composed of 3505 asthmatic women who had a total of 4593 pregnancies. Three hundred two patients with PIH and 3013 control subjects were identified. Compared with nonuse, inhaled SABA use during pregnancy was significantly associated with a reduced risk of PIH (adjusted rate ratios: >0-3 doses/week, 0.62 (95% CI, 0.44-0.87); > 3-10 doses/week, 0.51 (95% CI, 0.34-0.79); and >10 doses/week, 0.48 (95% CI, 0.30-0.75)). CONCLUSIONS: To our knowledge, this is the first study reporting that inhaled SABA use during pregnancy is associated with a reduced risk of PIH. Potential explanations include pharmacologic and physiological effects or residual confounding. CLINICAL IMPLICATIONS: These results increase the evidence about the safety of inhaled SABAs in this population, although they should not undervalue the importance of maintaining good control of asthma symptoms.     

False-Positive β-Galactosidase Staining in Osteoclasts by Endogenous Enzyme: Studies in Neonatal and Month-Old Wild-Type Mice

Escherichia coli β-galactosidase (β-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by β-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined β-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). β-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using β-gal as a marker gene.     

Correlation between baseline femoral neck marrow status and the development of femoral head osteonecrosis in corticosteroid-treated patients: a longitudinal study by MR imaging

OBJECTIVE: To test the hypothesis that the development of corticosteroid (CS)-associated femoral head osteonecrosis (ON) is influenced by baseline femoral neck marrow status. PATIENTS AND METHODS: The population consisted of 20 untreated patients with a newly diagnosed rheumatic disease in whom a standardized CS regimen was planned. Before CS treatment, baseline femoral neck marrow status was determined by magnetic resonance (MR) imaging on T1-weighted images (proportion of surface area of femoral neck and intertrochanteric area occupied by fatty marrow; index of marrow conversion [IMC]) and on a quantitative MR sequence (bulk T1 values of femoral head and neck). The presence of ON was assessed by coronal T1-weighted MR images of the hips at 6 and 12 months. RESULTS: None of the patients suffered from ON at baseline. Four patients (20%) developed bilateral femoral head ON at 6 months. The mean percentage of fat marrow in the femoral neck before treatment was significantly higher in ON-positive than in ON-negative patients (p=0.0025). The mean baseline femoral neck IMC value, which parallels the degree of red to yellow marrow conversion, was higher in ON-positive than in ON-negative patients (p=0.089). The mean baseline bulk T1 value of the femoral neck (but not of the femoral head), which inversely correlates with the amount of fat marrow, was significantly shorter in ON-positive than in ON-negative patients (p=0.0298). CONCLUSION: The development of CS-associated femoral head ON is correlated with a high fat content in the proximal femur before CS therapy.     

Efficacy of Imagery Rehearsal Therapy and Cognitive Behavioral Therapy in Sexual Assault Victims With Posttraumatic Stress Disorder: A Randomized Controlled Trial

Sleep disturbances are common among sexual assault victims with posttraumatic stress disorder (PTSD), but cognitive behavioral therapy (CBT) for PTSD does not directly address sleep‐related symptoms. Trauma‐related sleep disturbances are associated with more impairment and contribute to the maintenance of PTSD. In this study, we evaluated the efficacy of a combination of CBT and nightmare therapy (imagery rehearsal therapy; IRT) compared to CBT alone for the treatment of PTSD. We recruited 42 adult victims of sexual assault who were suffering from PTSD and randomly assigned them to either the experimental (IRT + CBT) or control condition (waiting period followed by CBT). After CBT, both groups demonstrated significant decreases in nighttime symptoms (except nightmare frequency) and PTSD symptoms and showed improvements in functional impairment and mental health, ds = 0.13–0.83, ps = .005–.008. Outcomes between the two groups did not differ significantly after CBT; however, we observed medium to medium‐large differences between the control group and experimental group in terms of nighttime symptoms, ds = 0.45–0.63. Although results did not clearly establish the superiority of IRT + CBT over CBT alone, they demonstrated that IRT yielded greater improvement in nighttime symptoms than the waiting period, ds = 0.72–1.13, ps = .006–.047 for all interaction effects. Findings suggest that targeting nightmares at the beginning of treatment for PTSD may yield rapid improvement in nighttime symptoms. This strategy could be useful for patients with time or resource constraints or those for whom nightmares are the primary complaint.     

Modifications of the organic and mineral fractions of dental tissues following conditioning by self-etching adhesives

Objectives

Our objective was to analyse the acid strengths and concentrations in contemporary self-etch adhesives and test whether the adhesion/decalcification concept functions the same way for all products.

Methods

The self-etching adhesives were dissolved in a 50% water-ethanol solvent, these were reacted with biological apatite (HA) in the form of powder of human dentine in order to quantify calcium release and study the reaction products as a function of acid strengths and concentrations. The four self-etching adhesives investigated were AdheSE One (Ivoclar Vivadent, Schaan, Liechtenstein), Adper Easy Bond (3 M ESPE, St Paul, MN, USA), Optibond All-In-One (KERR, Orange, CA, USA), Xeno V (Dentsply De Trey, Konstanz, Germany).

Results

Acid concentrations were found to span the range from 1 to 2 mmol/l, and the acid dissociation constants varied between apparent pKa values of 3.4 and 4.2. The pH values changed with time from values near 2.8 to 3.6, confirming the buffering action of HA. The stronger acids dissolved more calcium ions but left less organic matter attached to the tissue particles. Thermogravimetric and infrared analysis demonstrated that the weaker acids tended to bind to HA surfaces and increased significantly the organic to mineral ratios of the powders.

Conclusion

Self-etching adhesives can be differentiated and classified in two types: weak acids attach to the mineral phase and leach little calcium; strong acids bind to the calcium ions, demineralize more and tend to debond from the dentinal hard tissues by forming more soluble calcium salts.     

Mechanisms of the depot specificity of peroxisome proliferator-activated receptor gamma action on adipose tissue metabolism

In this study, we aimed to establish the mechanisms whereby peroxisome proliferator-activated receptor gamma (PPARgamma) agonism brings about redistribution of fat toward subcutaneous depots and away from visceral fat. In rats treated with the full PPARgamma agonist COOH (30 mg x kg(-1) x day(-1)) for 3 weeks, subcutaneous fat mass was doubled and that of visceral fat was reduced by 30% relative to untreated rats. Uptake of triglyceride-derived nonesterified fatty acids was greatly increased in subcutaneous fat (14-fold) and less so in visceral fat (4-fold), with a concomitant increase, restricted to subcutaneous fat only, in mRNA levels of the uptake-, retention-, and esterification-promoting enzymes lipoprotein lipase, aP2, and diacylglycerol acyltransferase 1. Basal lipolysis and fatty acid recycling were stimulated by COOH in both subcutaneous fat and visceral fat, with no frank quantitative depot specificity. The agonist increased mRNA levels of enzymes of fatty acid oxidation and thermogenesis much more strongly in visceral fat than in subcutaneous fat, concomitantly with a stronger elevation in O2 consumption in the former than in the latter. Mitochondrial biogenesis was stimulated equally in both depots. These findings demonstrate that PPARgamma agonism redistributes fat by stimulating the lipid uptake and esterification potential in subcutaneous fat, which more than compensates for increased O2 consumption; conversely, lipid uptake is minimally altered and energy expenditure is greatly increased in visceral fat, with consequent reduction in fat accumulation.     

Sequential development of Hodgkin's disease and CD30+ diffuse large B-cell lymphoma in a patient with MALT-type lymphoma: evidence of different clonal origin of single microdissected Reed-Sternberg cells

We observed in the same patient the development of a tonsil mucosa-associated lymphoid tissue-type lymphoma in 1994, a mediastinal Hodgkin's disease in 1998, and a colonic CD30+ anaplastic diffuse large B-cell lymphoma in 2000. A same-sized FR3-JH fragment was demonstrated by polymerase chain reaction, both at the level of total DNA and of single micromanipulated cells, showing monocytoid, Reed-Sternberg, or anaplastic morphology. Moreover, an identical IgH nucleotide sequence was detected in mucosa-associated lymphoid tissue-type lymphoma and colonic CD30+ anaplastic diffuse large B-cell lymphoma, whereas mediastinal Hodgkin's disease IgH rearrangement involved different VH and JH genes. CD30+ Reed-Sternberg and diffuse large B-cell lymphoma cells contained Epstein-Barr virus EBER sequences that were not observed at the level of mucosa-associated lymphoid tissue-type lymphoma. Therefore, Epstein-Barr virus infection may have played a role in diffuse large B-cell lymphoma transformation of mucosa-associated lymphoid tissue-type lymphoma and in the lymphomagenesis of Hodgkin's disease. In addition to their different clonal origin, Reed-Sternberg cells of Hodgkin's disease expressed a CD15+, CD20+ (rare cells), CD30+, Oct-2-, EBNA2-, LMP1+ phenotype, whereas anaplastic and Reed-Sternberg-like cells of diffuse large B-cell lymphoma were CD15-, CD20+, CD30+, Oct-2+, EBNA2+, and LMP1+. Interestingly, we also detected scattered CD30+ Epstein-Barr virus- large cells with prominent nucleoli in the initial tonsil mucosa-associated lymphoid tissue-type lymphoma, suggesting that these cells could be prone to Epstein-Barr virus infection and/or large cell transformation.