Muscular response to the first three months of deflazacort treatment in boys with Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) patients are often treated with glucocorticoids; yet their precise molecular action remains unknown. METHODS: We investigated muscle biopsies from nine boys with DMD (aged: 7,6±2,8 yrs.) collected before and after three months of deflazacort treatment and compared them to eight healthy boys (aged: 5,3±2,4 yrs.). mRNA transcripts involved in activation of satellite cells, myogenesis, regeneration, adipogenesis, muscle growth and tissue inflammation were assessed. Serum creatine kinase (CK) levels and muscle protein expression by immunohistochemistry of selected targets were also analysed. RESULTS: Transcript levels for ADIPOQ, CD68, CDH15, FGF2, IGF1R, MYF5, MYF6, MYH8, MYOD, PAX7, and TNFα were significantly different in untreated patients vs. normal muscle (p⟨0.05). Linear tests for trend indicated that the expression levels of treated patients were approaching normal values (p⟨0.05) following treatment (towards an increase; CDH15, C-MET, DLK1, FGF2, IGF1R, MYF5, MYF6, MYOD, PAX7; towards a decrease: CD68, MYH8, TNFα). Treatment reduced CK levels (p⟨0.05), but we observed no effect on muscle protein expression. CONCLUSIONS: This study provides insight into the molecular actions of glucocorticoids in DMD at the mRNA level, and we show that multiple regulatory pathways are influenced. This information can be important in the development of new treatments.

Ageing is associated with diminished muscle re-growth and myogenic precursor cell expansion early after immobility-induced atrophy in human skeletal muscle.

Recovery of skeletal muscle mass from immobilisation-induced atrophy is faster in young than older individuals, yet the cellular mechanisms remain unknown. We examined the cellular and molecular regulation of muscle recovery in young and older human subjects subsequent to 2 weeks of immobility-induced muscle atrophy. Retraining consisted of 4 weeks of supervised resistive exercise in 9 older (OM: mean age) 67.3, range 61-74 yrs) and 11 young (YM: mean age 24.4, range 21-30 yrs) males. Measures of myofibre area (MFA), Pax7-positive satellite cells (SCs) associated with type I and type II muscle fibres, as well as gene expression analysis of key growth and transcription factors associated with local skeletal muscle milieu, were performed after 2 weeks immobility (Imm) and following 3 days (+3d) and 4 weeks (+4wks) of retraining. OM demonstrated no detectable gains in MFA (vastus lateralis muscle) and no increases in number of Pax7-positive SCs following 4wks retraining, whereas YM increased their MFA (P < 0.05), number of Pax7-positive cells, and had more Pax7-positive cells per type II fibre than OM at +3d and +4wks (P < 0.05). No age-related differences were observed in mRNA expression of IGF-1Ea, MGF, MyoD1 and HGF with retraining, whereas myostatin expression levels were more down-regulated in YM compared to OM at +3d (P < 0.05). In conclusion, the diminished muscle re-growth after immobilisation in elderly humans was associated with a lesser response in satellite cell proliferation in combination with an age-specific regulation of myostatin. In contrast, expression of local growth factors did not seem to explain the age-related difference in muscle mass recovery.

Loss of ICAM-1 signaling induces psoriasin (S100A7) and MUC1 in mammary epithelial cells.

Psoriasin (S100A7), a member of the S100 gene family, is highly expressed in high-grade comedo ductal carcinoma in situ (DCIS), with a higher risk of local recurrence. Psoriasin is, therefore, a potential biomarker for DCIS with a poor prognosis. High-grade DCIS is characterized by a high proliferation rate and crowded cells, consequently, lose contact with the extracellular matrix. The aim of this study was, therefore, to elucidate the involvement of adhesion signals in the regulation of psoriasin. Protein expression was evaluated by Western blotting, flow cytometry, and immunohistochemistry, and using breast carcinoma SAGE databases available from the CGAP website. Intercellular adhesion molecule 1 (ICAM-1) was down-regulated in MCF10A cells using short hairpin RNA. We found a significant negative correlation between the expression of ICAM-1 and psoriasin, and a positive correlation between psoriasin and MUC1 in normal and DCIS SAGE libraries. In a cluster analysis of 34 adhesion molecules and 20 S100 proteins, we showed that SAGE libraries expressing the S100 proteins-psoriasin, calgranulin-A, and calgranulin-B-clustered together. Interestingly, the expression of all the three proteins correlated strongly to the oncogenic MUC1. We confirmed the negative correlation between ICAM-1 and psoriasin/MUC1, when normal and breast cancer cells were cultured in suspension and on collagen, respectively. The down-regulation of ICAM-1 by short hairpin RNA in MCF10A cells led to the induction of psoriasin, calgranulin-A, calgranulin-B, and MUC1, and we demonstrated that these up-regulations were not ROS dependent. By blocking the phospholipase C (PLC)-IP3 pathway in these cells, we showed that the induction of psoriasin diminished. The results suggest that psoriasin is an intracellular calcium-dependent target of the PLC pathway. Our findings suggest that the down-regulation of ICAM-1 in mammary epithelial cells may contribute both to the high expression of psoriasin seen in some high-grade DCIS tumors and to the induction of MUC1.

Elevation of serum epidermal growth factor and interleukin 1 receptor antagonist in active psoriasis vulgaris.

BACKGROUND: Psoriatic plaques present a complex expression profile, including high levels of cytokines, chemokines and growth factors. Circulating cytokines have been suggested to reflect the activation status of the inflammatory process. OBJECTIVES: To analyse 20 cytokines, chemokines and growth factors in 14 patients with psoriasis vulgaris at the start and during the course of ultraviolet B treatment. METHODS: A multiplex cytokine assay was used. RESULTS: We identified increased serum levels of epidermal growth factor (EGF) (mean 323 vs. 36·6 pg mL⁻¹, P = 0·0001), interleukin (IL)-1 receptor antagonist (mean 39·1 vs. 14·6 pg mL⁻¹, P = 0·02) and tumour necrosis factor-α (mean 7·5 vs. 4·5 pg mL⁻¹, P = 0·04) at baseline in patients with psoriasis compared with matched controls. None of these cytokines was correlated to the severity of the disease (Psoriasis Area and Severity Index) or decreased with phototherapy, suggesting that sources other than lesional skin contribute to the production of these cytokines. Using cluster analysis, we observed coordinate upregulation of EGF, IL-6, macrophage inflammatory protein-1ß and vascular endothelial growth factor. CONCLUSIONS: The sustained high expression of inflammatory circulating cytokines is a potential mechanism linking psoriasis with its extracutaneous comorbidities.

Cluster analysis of S100 gene expression and genes correlating to psoriasin (S100A7) expression at different stages of breast cancer development.

Gene expression patterns in ductal carcinoma in situ (DCIS) and invasive and metastatic breast tumors have been determined using serial analysis of gene expression (SAGE). The purpose of this approach was to identify biologically and clinically meaningful subgroups of DCIS with a high risk of progression to invasive disease. The analyses have led to the identification of several differentially expressed genes, such as HIN-1, dermcidin and S100A7 (psoriasin). The aim of the present study was further to delineate the expression profile of S100 genes using information from 22 breast epithelial SAGE libraries. We demonstrated the down-regulation of S100A6 and S100A10 in breast cancer, irrespective of pathological stage. S100P and S100Z were both up-regulated in cancer; whereas S100A7, S100A8 and S100A9 were strongly up-regulated only in DCIS. The hierarchical clustering of S100 gene expression in these 22 libraries revealed two major groups with distinguishable S100 gene expression profiles. One of them was characterized by the high concomitant expression of S100A7, S100A8 and S100A9. Using SAGE informatics, we found 21 genes with a high positive correlation to S100A7 expression in libraries representing different categories of tissues archived at SAGE Genie, suggesting a function of psoriasin that is not tissue specific. Like S100A7, several of these genes displayed cation-binding properties. We also report the strong correlation in the breast epithelial SAGE libraries between the expression of S100A7 and genes reported as being up-regulated in DCIS, as well as in the inflammatory skin disorder, psoriasis; including RGS5, UPK1A, TMPRSS3, S100A9, p53, SCCA1, SCCA2 and KRT17.

Expression of cholecystokinin, enkephalin, galanin and neuropeptide Y is markedly changed in the brain of the megencephaly mouse.

Megencephaly, enlarged brain, is a major sign in several human neurological diseases. The mouse model for megencephaly (mceph/mceph) has an enlarged brain, presumably due to brain cell hypertrophy, and exhibits neurological and motor disturbances with seizure-like activity, as well as disturbances in the insulin-like growth factor system. Here, we report that expression of the neuropeptides cholecystokinin, enkephalin, galanin and neuropeptide Y is dramatically changed in mceph/mceph brains compared to wild type, as revealed by in situ hybridization and immunohistochemistry. The changes were confined to discrete brain regions and occurred in a parallel fashion for peptides and their transcripts. For cholecystokinin, mceph/mceph brains had region-specific up- and down-regulations in several layers of the hippocampal formation and increased levels in, especially ventral, cortical regions. Enkephalin messenger RNA expression was up-regulated in the dentate gyrus granular layer and in ventral cortices, but down-regulated in the CA1 pyramidal layer. Enkephalin-like immunoreactivity was elevated in mossy fibers of the hippocampus and the ventral cortices. Galanin expression was increased in several layers and interneurons of the hippocampal formation, as well as in ventral cortices. Galanin-like immunoreactivity was reduced in nerve terminals in the forebrain. Neuropeptide Y expression was increased in the hippocampal formation and ventral cortices. Whether the mainly increased peptide levels contribute to the excessive growth of the brain or represent a consequence of this growth and/or of the neurological and motor disturbances remains to be elucidated.

Primary megalencephaly at birth and low intelligence level.

OBJECTIVES: To evaluate the association between primary megalencephaly (PMG) at birth and psychosensory conditions and to determine mother-child similarity for PMG at birth. BACKGROUND: PMG is defined as a head circumference (HC) above the 98th percentile that most likely is due to brain enlargement and is not secondary to disease. Previously, PMG in children was reported to be associated with learning disabilities. Contradictory results regarding an association between PMG and intelligence in children exist. Many believe PMG expressed in childhood and adulthood may be inherited. METHODS: Birth records on HC from 144,273 boys, of whom 732 had PMG, were linked to data concerning intelligence level, mental retardation, and impairment of vision and hearing. A potential association between PMG at birth and mental retardation was also examined in 3,204 mentally retarded boys and girls. Parent-offspring similarity for PMG at birth was determined in 13,585 mother-child pairs. RESULTS: PMG was significantly associated with low intelligence level (odds ratio, 1.32; 95% confidence interval [CI], 1.11 to 1.38). The estimated odds ratio for mental retardation in PMG cases versus controls was 1.31 (95% CI, 0.80 to 2.02). There were no differences in frequencies of vision and hearing impairments between PMG cases and controls. Associations between mother's and child's birth weight-normalized HC (r = 0.14; p<0.0001) and PMG (odds ratio, 2.55; CI, 2.00 to 3.25) were found, supporting a multifactorial inheritance of PMG. CONCLUSIONS: PMG at birth is a risk factor for low intelligence level but not for vision and hearing impairments. The heritability of HC and PMG is moderate.

The megencephaly mouse has disturbances in the insulin-like growth factor (IGF) system.

Megencephaly, enlarged brain, is a major sign in several human neurological diseases. The mouse model for megencephaly, mceph/mceph, has an enlarged brain and a lowered body weight. In addition, it displays several neurological and motoric disturbances. Previous studies suggest that the brain enlargement results from hypertrophy of the brain cells, rather than hyperplasia. No structural abnormalities, edema or increased myelination have been found. In this study, a major imbalance in the mRNA expression of molecules in the insulin-like growth factor (IGF) system was found in brains of 9-10 weeks old mceph/mceph mice compared to +/+ wild-type mice. In mceph/mceph brains, we found upregulation of IGF binding proteins (BP)-2, -4, -5, and -6 mRNA, the regulating hormone transforming growth factor (TGF)beta1 mRNA and also a local downregulation of IGFBP-5 mRNA compared to wild-type brains by in situ hybridization. The altered expression of these mRNA species is colocalized in cerebral cortex, hippocampus, amygdala and piriform/entorhinal cortex. The mceph/mceph mice express less of the myelin component proteolipid protein (PLP) mRNA in corpus callosum. No expression difference of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in brain or IGF system components in liver was found between mceph/mceph and wild-type mice. These data suggest that the IGF system has an important role in the excessive growth of the mceph/mceph brains.

Deletions of the N-terminal regions of the human melanocortin receptors.

The non-homologous N-terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and analysed by ligand binding. The results show that 27, 25, 28, and 20 amino acids could be deleted from the N terminus of the human MC1, MC3, MC4 and MC5 receptors, respectively, including all potential N-terminal glycosylation sites in the MC1 and the MC4 receptors, without affecting ligand binding or expression levels. The results indicate that the N-terminal regions of the human MC1, MC3, MC4 and MC5 receptors, do not play an important role for the ligand binding properties of these receptors.

Effect of transurethral microwave thermotherapy on serum concentrations of total and free prostate-specific antigen.

This study includes 15 men between 48 and 81 years of age with treatment-requiring symptomatic benign prostatic hyperplasia (BPH). No patient with a urinary infection or prostate cancer was included. The serum concentrations of prostate-specific antigen (PSA), total as well as "free", i.e. noncomplex bound, were repeatedly determined before and up to 3 months after transurethral microwave thermotherapy (TUMT) of the prostate. After TUMT we found significant rises of the ratios of the serum total PSA concentration/prostate volume (up to 55-fold, mean 13.1) and of the serum free PSA concentration/ prostate volume (up to 26-fold, mean 10.2). The highest values for the serum total PSA concentration/prostate volume ratio were found on the first day after TUMT. Maximal serum free PSA concentration/prostate volume ratio occurred 3-6 h after treatment. The serum concentration/prostate volume ratios for total and free PSA returned to normal within 3 months. The results indicate that PSA determinations might be used as effect indicators of TUMT.

Biocontrol of Mold Growth in High-Moisture Wheat Stored under Airtight Conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Volume 61, no. 3, p. 1029, Table 1, boxhead over columns 2 to 4: "(CFU/plate)" should read "(log CFU/plate)." FIG. 1 [This corrects the article on p. 1027 in vol. 61.].

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.

A modified palatal arch for treatment of unilateral functional cross-bite in the primary dentition.

A modified palatal arch, based on the classical principle of the safety pin and aimed at bilateral expansion in cases with a unilateral functional cross-bite, is described. It is illustrated with case reports.

Ferrous sulphate gels for determination of absorbed dose distributions using MRI technique: basic studies.

Two gels have been found to be suitable to load with ferrous sulphate solution. In these soft tissue equivalent phantoms, the absorbed dose distribution can be measured after irradiation in clinically used MR imaging equipment. The present studies were carried out using a 0.25 T NMR analyser without imaging properties. A ferrous sulphate solution, 0.05 M with respect to sulphuric acid, can be gelled with 4% gelatin to give a dosemeter which has a response which is linearly correlated (r = 0.998) with the absorbed dose in the interval 0-40 Gy. Ferrous sulphate solution can also be gelled with 1% agarose, but this gel has to be purged with oxygen to obtain a linear relationship (r = 0.997) in the same absorbed dose interval. The ferrous sulphate loaded gels have a sensitivity which is a factor of 2.2 or 4.0 times higher for gelatin and agarose, respectively, than the ordinary dosemeter solution. Because the standard deviation of background measurements is higher for the gels than for the dosemeter solution, the minimum detectable absorbed dose is about the same, or 1.0 Gy, for the two gels and the dosemeter solution. The sensitivity of the ferrous sulphate loaded gels shows no dependence on dose rate if the mean dose rate and the absorbed dose per pulse are within the limits normally used by accelerators for radiotherapy.