Prevalence and predictors of anaemia in Romanian infants 6-23 months old.

BACKGROUND: Anaemia is a public health problem that can lead to a variety of detrimental effects on physical and neurodevelopment in young children. The present study explored the epidemiology of anaemia among infants in Romania, identified risk factors and created a model for predicting it. METHODS: Data from 1532 infants aged 6-24 months were selected from a larger nationally representative cross-sectional survey. Demographic predictor variables and haemoglobin concentration were extant variables in the data set. Multiple logistic regression was used to determine the best predictors of anaemia. RESULTS: Overall, 46% of 6-24 month olds in the sample had anaemia (Hb < 11.0 g/dl). A variety of risk factors were associated with significantly greater odds of anaemia, but a five-factor model best predicted it (67.9% accuracy). These predictors included being male, living in a rural area, being third born or later, being a Hungarian and living in the South, South-West or West region of Romania. CONCLUSIONS: While data indicate a modest decrease in anaemia from earlier Romanian studies, it remains a significant problem. Models like this one have the potential to improve identification and treatment of anaemia in young children.

Clinical characterisation of Becker muscular dystrophy patients predicts favourable outcome in exon-skipping therapy.

OBJECTIVE: Duchenne and Becker muscular dystrophy (DMD/BMD) are both caused by mutations in the DMD gene. Out-of-frame mutations in DMD lead to absence of the dystrophin protein, while in-frame BMD mutations cause production of internally deleted dystrophin. Clinically, patients with DMD loose ambulance around the age of 12, need ventilatory support at their late teens and die in their third or fourth decade due to pulmonary or cardiac failure. BMD has a more variable disease course. The disease course of patients with BMD with specific mutations could be very informative to predict the outcome of the exon-skipping therapy, aiming to restore the reading-frame in patients with DMD. METHODS: Patients with BMD with a mutation equalling a DMD mutation after successful exon skipping were selected from the Dutch Dystrophinopathy Database. Information about disease course was gathered through a standardised questionnaire. Cardiac data were collected from medical correspondence and a previous study on cardiac function in BMD. RESULTS: Forty-eight patients were included, representing 11 different mutations. Median age of patients was 43 years (range 6-67). Nine patients were wheelchair users (26-56 years). Dilated cardiomyopathy was present in 7/36 patients. Only one patient used ventilatory support. Three patients had died at the age of 45, 50 and 76 years, respectively. CONCLUSIONS: This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a 'skipped' DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.

Dystrophin levels and clinical severity in Becker muscular dystrophy patients.

OBJECTIVE: Becker muscular dystrophy (BMD) is characterised by broad clinical variability. Ongoing studies exploring dystrophin restoration in Duchenne muscular dystrophy ask for better understanding of the relation between dystrophin levels and disease severity. We studied this relation in BMD patients with varying mutations, including a large subset with an exon 45-47 deletion. METHODS: Dystrophin was quantified by western blot analyses in a fresh muscle biopsy of the anterior tibial muscle. Disease severity was assessed using quantitative muscle strength measurements and functional disability scoring. MRI of the leg was performed in a subgroup to detect fatty infiltration. RESULTS: 33 BMD patients participated. No linear relation was found between dystrophin levels (range 3%-78%) and muscle strength or age at different disease milestones, in both the whole group and the subgroup of exon 45-47 deleted patients. However, patients with less than 10% dystrophin all showed a severe disease course. No relation was found between disease severity and age when analysing the whole group. By contrast, in the exon 45-47 deleted subgroup, muscle strength and levels of fatty infiltration were significantly correlated with patients' age. CONCLUSIONS: Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45-47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.

Spatial transcriptomics reveal markers of histopathological changes in Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The identification of genes and cell types driving tissue remodeling is a key step to developing effective therapies. Here we use spatial transcriptomics in two Duchenne muscular dystrophy mouse models differing in disease severity to identify gene expression signatures underlying skeletal muscle pathology and to directly link gene expression to muscle histology. We perform deconvolution analysis to identify cell types contributing to histological alterations. We show increased expression of specific genes in areas of muscle regeneration (Myl4, Sparc, Hspg2), fibrosis (Vim, Fn1, Thbs4) and calcification (Bgn, Ctsk, Spp1). These findings are confirmed by smFISH. Finally, we use differentiation dynamic analysis in the D2-mdx muscle to identify muscle fibers in the present state that are predicted to become affected in the future state.

[Syndromic surveillance of Influenza-like illness in primary care: a complement to the sentinel surveillance network for periods of increased incidence of Influenza]. / Vigilancia sindrómica de la gripe en atención primaria, un instrumento complementario a las redes centinelas para períodos de elevada incidencia de gripe.

OBJECTIVE: Epidemiological data on influenza is essential for resource management when the incidence of the disease in the population is very high, but not easily available in real-time. The objective of this study was to evaluate the use of a syndromic surveillance system for influenza-like illness in Primary Care (ILIsPC) and assess its level of agreement with the epidemiological data from the Influenza Sentinel Network. LOCALIZATION: Health centres and deputising medical services in the Balearic Islands. PARTICIPANTS: Data from 122 epidemiological weeks for each system were included. MAIN MEASURES: Data from January 1, 2007 to January 31, 2010 were compared. ILIsPC rates were obtained from the diagnoses registered in electronic health records of Primary Care clinics and deputising medical services. Data from Sentinel Network were obtained from weekly epidemiological reports. Intraclass correlation coefficient was calculated and Bland - Altman plot constructed. RESULTS: There was good agreement between both measures, with an intraclass correlation coefficient of 0.88 (95% CI: 0.83-0.91). After constructing a Bland-Altman plot, the precision between both rates was greater during the periods of the highest influenza incidence. CONCLUSIONS: We believe that the syndromic surveillance system ILIsPC, provides access to very useful data in real-time, especially during periods of high influenza incidence, such as during epidemics or the recent pandemic.

Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting.

Reduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting. VIDEO ABSTRACT.

Natural disease history of the dy2J mouse model of laminin α2 (merosin)-deficient congenital muscular dystrophy.

Merosin deficient congenital muscular dystrophy 1A (MDC1A) is a very rare autosomal recessive disorder caused by mutations in the LAMA2 gene leading to severe and progressive muscle weakness and atrophy. Although over 350 causative mutations have been identified for MDC1A, no treatment is yet available. There are many therapeutic approaches in development, but the lack of natural history data of the mouse model and standardized outcome measures makes it difficult to transit these pre-clinical findings to clinical trials. Therefore, in the present study, we collected natural history data and assessed pre-clinical outcome measures for the dy2J/dy2J mouse model using standardized operating procedures available from the TREAT-NMD Alliance. Wild type and dy2J/dy2J mice were subjected to five different functional tests from the age of four to 32 weeks. Non-tested control groups were taken along to assess whether the functional test regime interfered with muscle pathology. Respiratory function, body weights and creatine kinase levels were recorded. Lastly, skeletal muscles were collected for further histopathological and gene expression analyses. Muscle function of dy2J/dy2J mice was severely impaired at four weeks of age and all mice lost the ability to use their hind limbs. Moreover, respiratory function was altered in dy2J/dy2J mice. Interestingly, the respiration rate was decreased and declined with age, whereas the respiration amplitude was increased in dy2J/dy2J mice when compared to wild type mice. Creatine kinase levels were comparable to wild type mice. Muscle histopathology and gene expression analysis revealed that there was a specific regional distribution pattern of muscle damage in dy2J/dy2J mice. Gastrocnemius appeared to be the most severely affected muscle with a high proportion of atrophic fibers, increased fibrosis and inflammation. By contrast, triceps was affected moderately and diaphragm only mildly. Our study presents a complete natural history dataset which can be used in setting up standardized studies in dy2J/dy2J mice.

Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in the absence of functional protein. In the majority of cases these are out-of-frame deletions that disrupt the reading frame. Several attempts have been made to restore the dystrophin mRNA reading frame by modulation of pre-mRNA splicing with antisense oligonucleotides (AOs), demonstrating success in cultured cells, muscle explants, and animal models. We are preparing for a phase I/IIa clinical trial aimed at assessing the safety and effect of locally administered AOs designed to inhibit inclusion of exon 51 into the mature mRNA by the splicing machinery, a process known as exon skipping. Here, we describe a series of systematic experiments to validate the sequence and chemistry of the exon 51 AO reagent selected to go forward into the clinical trial planned in the United Kingdom. Eight specific AO sequences targeting exon 51 were tested in two different chemical forms and in three different preclinical models: cultured human muscle cells and explants (wild type and DMD), and local in vivo administration in transgenic mice harboring the entire human DMD locus. Data have been validated independently in the different model systems used, and the studies describe a rational collaborative path for the preclinical selection of AOs for evaluation in future clinical trials.

Platelet activation and red blood cell phosphatidylserine exposure evaluated by flow cytometry in patients with Behçet's disease: are they related to thrombotic events?

Behçet's disease (BD) is associated with an increased risk of venous and arterial thromboses that are associated with morbidity and mortality increase, although the mechanisms are not well established. In the present study, we used whole blood cytometry to determine the exposure of CD62 on the surface of platelets and the expression of phosphatidylserine (PS) on the surface of circulating red blood cells. Microparticle and microaggregate formation from platelets were also determined in a well-classified group of 72 patients (39 males, 33 females, aged 46.5 +/- 12.5 years) with BD, in comparison with a well-matched control group of 72 healthy volunteers. Results showed no differences in the above-mentioned parameters when BD patients and controls were compared. However, when we compared BD patients with/without thrombosis using these parameters, there were significant differences between both groups. BD patients with previous thrombosis had a higher percentage of circulating CD62-positive platelets and a higher number of circulating microaggregates than those without thrombosis, suggesting that platelet activation may be involved in the development of thrombotic events in these patients.

Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F.

Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice.

Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne.

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients.

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides.

Antisense oligonucleotides (AONs) can be used to correct the disrupted reading frame of Duchenne muscular dystophy patients (DMD). We have a collection of 121 AONs, of which 79 are effective in inducing the specific skipping of 38 out of the 79 different DMD exons. All AONs are located within exons and were hypothesized to act by steric hindrance of serine-arginine rich (SR) protein binding to exonic splicing enhancer (ESE) sites. Indeed, retrospective in silico analysis of effective versus ineffective AONs revealed that the efficacy of AONs is correlated to the presence of putative ESE sites (as predicted by the ESEfinder and RESCUE-ESE software). ESE predicting software programs are thus valuable tools for the optimization of exon-internal antisense target sequences.

Weaning induces NOS-2 expression through NF-kappaB modulation in the lactating mammary gland: importance of GSH.

At the end of lactation the mammary gland undergoes involution, a process characterized by apoptosis of secretory cells and tissue remodelling. To gain insight into this process, we analysed the gene expression profile by oligonucleotide microarrays during lactation and after forced weaning. Up-regulation of inflammatory mediators and acute-phase response genes during weaning was found. Expression of IkappaBalpha (inhibitory kappaBalpha), a protein known to modulate NF-kappaB (nuclear factor-kappaB) nuclear translocation, was significantly up-regulated. On the other hand, there was a time-dependent degradation of IkappaBalpha protein levels in response to weaning, suggesting a role for NF-kappaB. Furthermore, we have demonstrated, using chromatin immunoprecipitation assays, binding of NF-kappaB to the NOS-2 (inducible nitric oxide synthase) promoter at the early onset of events triggered during weaning. The three isoforms of NOS are constitutively present in the lactating mammary gland; however, while NOS-2 mRNA and protein levels and, consequently, NO production are increased during weaning, NOS-3 protein levels are diminished. Western blot analyses have demonstrated that protein nitration is increased in the mammary gland during weaning, but this is limited to a few specific tyrosine-nitrated proteins. Interestingly, inhibition of GSH synthesis at the peak of lactation partially mimics these findings, highlighting the role of NO production and GSH depletion during involution.

Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.

Studying the role of dystrophin-associated proteins in influencing Becker muscular dystrophy disease severity.

Becker muscular dystrophy is characterized by a variable disease course. Many factors have been implicated to contribute to this diversity, among which the expression of several components of the dystrophin associated glycoprotein complex. Together with dystrophin, most of these proteins anchor the muscle fiber cytoskeleton to the extracellular matrix, thus protecting the muscle from contraction induced injury, while nNOS is primarily involved in inducing vasodilation during muscle contraction, enabling adequate muscle oxygenation. In the current study, we investigated the role of three components of the dystrophin associated glycoprotein complex (beta-dystroglycan, gamma-sarcoglycan and nNOS) and the dystrophin homologue utrophin on disease severity in Becker patients. Strength measurements, data about disease course and fresh muscle biopsies of the anterior tibial muscle were obtained from 24 Becker patients aged 19 to 66. The designation of Becker muscular dystrophy in this study was based on the mutation and not on the clinical severity. Contrary to previous studies, we were unable to find a relationship between expression of nNOS, beta-dystroglycan and gamma-sarcoglycan at the sarcolemma and disease severity, as measured by muscle strength in five muscle groups and age at reaching several disease milestones. Unexpectedly, we found an inverse correlation between utrophin expression at the sarcolemma and age at reaching disease milestones.

Amino, chloromethyl and acetal-functionalized latex particles for immunoassays: a comparative study.

Latex particles with different functionalized surface groups (amino, acetal and chloromethyl) for the covalent linking of protein molecules were synthesized and characterized. Immunopurified anti-ferritin antibodies were then covalently coupled with a mean efficiency rate (protein covalently bound to latex particles with respect to the total amount of protein added) of 60%. The reagents developed were applied to the measurement of serum ferritin concentration in a turbidimetric procedure, showing a good measuring range and a lowest detection limit of 3.5 ng/ml in the case of the amino-modified particles. These immunological reagents were compared with a commercial nephelometric method, showing a good linear correlation in all cases but no transferability in the acetal and chloromethyl latex with additional carboxyl groups, probably due to interference with other serum components. The differences among latex found in this study indicate that it would be necessary to optimize the assay conditions for each type of particle, in order to achieve a maximum immunoreactivity.

Automated determination of apolipoproteins A-I and B with an improved immunoturbidimetric assay using Boehringer Mannheim and Hitachi analyzer systems.

Results from an evaluation of immunoturbidimetric methods (Tina-quant) for apolipoprotein A-I (apo AI) and apolipoprotein B (apo B), are presented and compared with results from six other commercial immunoassays. These apo AI and B procedures are fully automated on the Boehringer Mannheim (BM) and Hitachi 704 and 717 analyzers and use undiluted serum. The methods provide an analytical range from 0.002 to 2.3 g/L for apo AI and from 0.003 to 2.2 g/L for apo B and are precise with maximal CVs of 4%. Neither hemoglobin nor intralipid interfered with the assays. Bilirubin concentrations higher than 376 mumol/L for apo AI and 444 mumol/L for apo B, produce a negative interference. In the apo AI method a positive interference caused by rheumatoid factor was found for concentrations higher than 440 x 10(3) IU/L. All comparisons showed an excellent correlation (r > 0.93) with all methods, and slopes ranged from 1.01 to 1.25 for apo AI, and from 0.62 to 1.15 for apo B.

Rheumatoid arthritis and hepatitis C virus antibodies.

The prevalence of antibodies against the hepatitis C virus (anti-HCV) in patients seropositive for rheumatoid arthritis (RA) and suffering from this disease has been analyzed in comparison to a control group. Our results suggest that the high rate of prevalence found in RA patients is due to IgG interference in the measuring method used.

Malaria and Anopheles mosquitos in Malaysia.

Until today, malaria is still one of the most important diseases in Malaysia. This is because Malaysia is located within the equatorial zone with high temperatures and humidities, usually important for the transmission of malaria. The number of malaria cases were estimated to be around 300,000 before the launching of the Malaria Eradication Program (MEP). The program was successful in reducing the numbers progressively during the 1967-1982 period. During the period 1980-1991, the highest number of malaria cases recorded for the country was 65,283 in 1989 (16,902 in Peninsular Malaysia, 47,545 in Sabah and 836 in Sarawak) whilst the lowest was 22,218 (10,069 in Peninsular Malaysia, 11,290 in Sabah and 859 in Sarawak) in 1983. In Malaysia, there are 434 species of mosquitos, representing 20 genera. Of these, 75 species are Anopheles that comprise of 2 subgenus, i.e. Anopheles and Cellia. Of the 75 species, only 9 have been reported as vectors: An. maculatus, An balabacensis, An. dirus, An. letifer An. campestris, An. sundaicus, An. donaldi, An. leucophyrus and An. flavirostris. The behavior, seasonal abundance, biting activities and breeding sites of these species are discussed.