Analysis of point mutations in the SMN1 gene in SMA patients bearing a single SMN1 copy

Spinal muscular atrophy (SMA) is caused by homozygous deletion of the SMN1 gene in approximately 96% of cases. Four percent of SMA patients have a combination of the deletion or conversion on one allele and an intragenic mutation on the second one. We performed analysis of point mutations in a set of our patients with suspicion of SMA and without homozygous deletion of the SMN1 gene. A quantitative test determining SMN1 copy number (using real-time PCR and/or MLPA analysis) was performed in 301 patients and only 1 SMN1 copy was detected in 14 of them. When these 14 patients were screened for the presence of point mutations we identified 6 mutations, p.Y272C (in three patients) and p.T274I, p.I33IfsX6, and p.A188S (each in one case). The mutations p.I33IfsX6 and p.A188S were found in two SMAI patients and were not detected previously. Further, evaluation of the relationship between mutation type, copy number of the SMN2 gene and clinical findings was performed. Among our SMA patients with a SMN1 homozygous deletion, we found a family with two patients: the son with SMAII possesses 3 SMN2 copies and the nearly asymptomatic father has a homozygous deletion of SMN1 exon 7 and carries 4 SMN2 copies. Generally, our results illustrate that an increased SMN2 gene copy number is associated with a milder SMA phenotype.     

Effect of adenosine on the growth of human T-lymphocyte leukemia cell line MOLT-4

Adenosine has been observed to suppress the growth of MOLT-4 human leukemia cells in vitro. Changes in the cell cycle, especially increased percentage of cells in S phase, prolonged generation time, and induction of apoptosis at higher adenosine concentrations have been found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, reversed partially but significantly the adenosine-induced growth suppression. It follows from these results that the action of adenosine on the MOLT-4 cells comprises its cellular uptake and intracellular operation. These findings present new data on anticancer efficacy of adenosine.     

Ventilator-associated pneumonia: prevention is better than cure

Background: The prevention of ventilator Assisted Pneumonia (VAP), a hospital acquired infection, among intensive care patients is a major clinical challenge. It is a condition that is associated with high rates of morbidity, mortality, length of stay and hospital costs. Aim: The aim of this paper is to critically review the available literature and identify current evidence based nursing and medical interventions to support practitioners in preventing VAP in their patients. Methods: A literature search using keywords, including ‘ventilator‐associated pneumonia’ were entered into a search engine. A number of highly pertinent papers relevant to the aims of the review were identified, however only a small sample came from nursing journals. Only those papers, which discussed specific strategies for managing VAP were selected for analysis and inclusion in this review. Discussion: We identified a number of practical and evidence based strategies that nurses can incorporate into their practice to prevent VAP and to reduce its incidence. In addition, the introduction of newer techniques, advances in equipment and use of multidisciplinary care bundles can further support and improve the quality and delivery of safe patient care. Conclusion: Targeted strategies aimed at preventing VAP, should be implemented to improve patient outcome and reduce length of intensive care unit stay and costs. Front‐line critical care nurses need to understand the factors which place their patients at risk of developing VAP and, institute evidence‐based interventions that will compromise the patients’ survival and recovery.     

Atypical CLN2 with later onset and prolonged course: a neuropathologic study showing different sensitivity of neuronal subpopulations to TPP1 deficiency

This is the first neuropathology report of a male patient (born 1960-died 1975) with an extremely rare, atypical variant of CLN2 that has been diagnosed only in five families so far. The clinical history started during his preschool years with relatively mild motor and psychological difficulties, but with normal intellect and vision. Since age six there were progressive cerebellar and extrapyramidal symptomatology, amaurosis, and mental deterioration. Epileptic seizures were absent. The child died aged 15 years in extreme cachexy. Neuropathology revealed neurolysosomal storage of autofluorescent, curvilinear and subunit c of mitochondrial ATP synthase (SCMAS) rich material. The neuronal storage led to laminar neuronal depopulation in the cerebral cortex and to a practically total eradication of the cerebellar cortical neurons. The other areas of the central nervous system including hippocampus, which are usually heavily affected in classical forms of CLN2, displayed either a lesser degree or absence of neuronal storage, or storage without significant neuronal loss. Transformation of the stored material to the spheroid like perikaryal inclusions was rudimentary. The follow-up, after 30 years, showed heterozygous values of TPP1 (tripeptidylpeptidase 1) activity in the white blood cells of both parents and the sister. DNA analysis of CLN2 gene identified a paternal frequent null mutation c.622C > T (p.Arg208 X) in the 6th exon and a maternal novel mutation c.1439 T > G in exon 12 (p.Val480Gly). TPP1 immunohistochemistry using a specific antibody gave negative results in the brain and other organs. Our report supports the notion that the spectrum of CLN2 phenotypes may be surprisingly broad. The study revealed variable sensitivities in neuronal subpopulations to the metabolic defect which may be responsible for the variant's serious course.     

Epstein-Barr virus infection and risk of lymphoma: immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controls

Epstein-Barr Virus (EBV) is consistently associated with distinct lymphoproliferative malignancies and aberrant EBV antibody patterns are found in most EBV cancer patients. We evaluate the detection of an abnormal reactive serological pattern to EBV (ab_EBV) infection and the risk of lymphoma in a multicentric case-control study. Serum samples were collected at study entry from 1,085 incident lymphoma cases from Spain, France, Germany, Czech Republic, Italy and 1,153 age, sex and country matched controls. EBV immunoglobulin G (IgG) serostatus was evaluated through a peptide-based ELISA combining immunodominant epitopes of EBNA1 (BKRF1) and VCA-p18 (BFRF3). Further, immunoblot analysis was performed to evaluate distinct antibody diversity patterns to EBV early antigens (EA), besides EBNA1, VCA-p18, VCA-p40 (BdRF1) and Zebra (BZLF1). Patients with chronic active EBV infection and aberrant EBV activity were characterized as having an abnormal reactive pattern (ab_EBV). Ab_EBV was observed in 20.9% of 2,238 included subjects with an increased proportion of cases presenting ab_EBV as compared to the control population (23.9% vs. 18.0% p = 0.001). Ab_EBV positivity was a risk factor for all lymphomas combined (odds ratio [OR] = 1.42, 95% confidence interval [CI]=1.15-1.74), and specifically for chronic lymphocytic leukaemia (OR = 2.96, 95%CI = 2.22-3.95). Lower levels of ab_EBV were observed for follicular lymphoma (OR = 0.38, 95%CI = 0.15-0.98). EBV may be involved in a larger subset of lymphomas among clinically immunocompetent subjects than previously thought, probably explained by an underlying loss of immune control of EBV latent infection. Ab_EBV is a useful tool to explore EBV imbalances preceding or paralleling possible EBV associated oncogenic events.     

Candidate markers for the detection of hepatocellular carcinoma in low-molecular weight fraction of serum

Hepatocellular carcinoma (HCC) represents an important public health problem in Egypt where up to 90% of HCC cases are attributable to hepatitis C viral (HCV) infection. Serum alpha-fetoprotein is elevated in only approximately 60% of HCC patients. The development of effective markers for the detection of HCC could have an impact on cancer mortality and significant public health implications worldwide. The objective of our study was to assess six candidate markers for detection of HCC identified by mass spectrometric analysis of enriched serum. The study examined 78 HCC cases and 72 age- and gender-matched cancer-free controls recruited from the Egyptian population. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometric analysis of enriched low-molecular weight fraction of serum was used for identification of the candidate markers. Our analyses show that all six candidate markers are associated with HCC after adjustment for important covariates including HCV and hepatitis B viral infections. The marker candidates are independently predictive of HCC with areas under the receiver operating characteristic (AuROC) curve ranging from 63-93%. A combination of the six markers improves prediction accuracy to 100% sensitivity, 91% specificity and 98% AuROC curve in an independent test set of 50 patients. Two of the candidate markers were identified by sequencing as fragments of complement C3 and C4. In conclusion, a set of six peptides distinguished with high prediction accuracy HCC from controls in an Egyptian population with a high rate of chronic HCV infection. Further evaluation of these marker candidates for the diagnosis of HCC is needed.     

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1^−/− mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1^−/−;Wnt5a^−/− mice revealed a greater loss of Nurr1⁺ cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson’s disease.