Assessing drug effect from distributional data: A population approach with application to Duchenne Muscular Dystrophy treatment.

BACKGROUND AND OBJECTIVE: In Duchenne Muscular Dystrophy (DMD) treatment, muscle fiber size can be considered as an indicator of muscle health and function. In particular, the statistical distribution of fibers cross-sectional areas (CSAs) has been used as quantitative efficacy endpoint. For each patient, assessment of treatment effect relies on the comparison of pre- and post-treatment biopsies. Since biopsies provide "distributional data", i.e. empirical distributions of fibers CSA, the comparison must be carried out between the empirical pre- and post-treatment distributions. METHODS: Here, distributional fiber CSA data are analyzed by means of a hierarchical statistical model based on the population approach, considering both the single patient and the population level. RESULTS: The proposed method was used to assess the histological clinical effects of Givinostat, a compound under study for DMD treatment. At the single patient level, a two-component Gaussian mixture adequately represents pre- and post-treatment distributions of log-transformed CSAs; drug effect is described via a dose-dependent multiplicative increase of muscle fiber size. The single patient model was also validated via muscle composition data. At the patient population level, typical model parameters and inter-patient variabilities were obtained. CONCLUSIONS: The proposed methodological approach completely characterizes fiber CSA distributions and quantifies drug effect on muscle fiber size, both at the single patient and at the patient population level. This approach might be applied also in other contexts, where outcomes measured in terms of distributional data are to be assessed.

Laboratory-scale investigation on the role of microalgae towards a sustainable treatment of real municipal wastewater.

Engineered microalgal-bacteria consortia are an attractive solution towards a low-cost and sustainable wastewater treatment that does not rely on artificial mechanical aeration. In the research conducted for this study, a bench-scale photo-sequencing-batch reactor (PSBR) was operated without external aeration. A spontaneous consortium of microalgae and bacteria was developed in the PSBR at a concentration of 0.8-1.7 g TSS/L. The PSBR ensured removal efficiency of 85 ± 8% for chemical oxygen demand (COD) and 98 ± 2% for total Kjeldahl nitrogen (TKN). Nitrogen balance revealed that the main mechanisms for TKN removal was autotrophic nitrification, while N assimilation and denitrification accounted for 4% and 56%, respectively. The development of dense microalgae-bacteria bioflocs resulted in good settleability with average effluent concentration of 16 mgTSS/L. The ammonium removal rate was 2.9 mgN L-1 h-1, which corresponded to 2.4 mgN gTSS-1 h-1. Although this specific ammonium removal rate is similar to activated sludge, the volumetric rate is lower due to the limited total suspended solids (TSS) concentration (three times less than activated sludge). Therefore, the PSBR footprint appears less competitive than activated sludge. However, ammonium was completely removed without artificial aeration, resulting in a very cost-effective process. Only 50% of phosphorus was removed, suggesting that further research on P uptake is needed.

Enhanced nitrogen removal and energy saving in a microalgal-bacterial consortium treating real municipal wastewater.

The optimization of total nitrogen removal from municipal wastewater was investigated in a laboratory-scale photo-sequencing batch reactor (PSBR) operated with a mixed microalgal-bacterial consortium spontaneously acclimatized to real wastewater. No external aeration was provided in the PSBR to reduce energy consumption: oxygen was only supplied by the microalgal photosynthesis. The enhancement of total nitrogen removal was achieved through: (1) feeding of wastewater in the dark phase to provide readily biodegradable COD when oxygen was not produced, promoting denitrification; (2) intermittent use of the mixer to favor simultaneous nitrification-denitrification inside the dense flocs and to achieve 41% energy saving with respect to continuous mixing. Efficient COD removal (86 ± 2%) was observed, obtaining average effluent concentrations of 37 mg/L and 22 mg/L of total COD and soluble COD, respectively. TKN removal was 97 ± 3%, with an average effluent concentration of 0.5 ± 0.7 mg NH4 +-N/L. Assimilation of nitrogen by heterotrophic bacteria accounted only for 20% of TKN removal, whilst the major part of TKN was nitrified. In particular, the nitrification rate was 1.9 mgN L-1 h-1 (specific rate 2.4 mgN gTSS-1 h-1), measured with dissolved oxygen near zero, when the oxygen demand was higher than the oxygen produced by photosynthesis. Total nitrogen of 6.3 ± 4.4 mgN/L was measured in the effluent after PSBR optimization.

The Vici syndrome protein EPG5 regulates intracellular nucleic acid trafficking linking autophagy to innate and adaptive immunity.

Vici syndrome is a human inherited multi-system disorder caused by recessive mutations in EPG5, encoding the EPG5 protein that mediates the fusion of autophagosomes with lysosomes. Immunodeficiency characterized by lack of memory B cells and increased susceptibility to infection is an integral part of the condition, but the role of EPG5 in the immune system remains unknown. Here we show that EPG5 is indispensable for the transport of the TLR9 ligand CpG to the late endosomal-lysosomal compartment, and for TLR9-initiated signaling, a step essential for the survival of human memory B cells and their ultimate differentiation into plasma cells. Moreover, the predicted structure of EPG5 includes a membrane remodeling domain and a karyopherin-like domain, thus explaining its function as a carrier between separate vesicular compartments. Our findings indicate that EPG5, by controlling nucleic acids intracellular trafficking, links macroautophagy/autophagy to innate and adaptive immunity.

The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines.

OBJECTIVES: Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines. MATERIALS AND METHODS: A549 and CALU1 cells' viability was assessed by MTT and trypan blue exclusion assays. Cell proliferation was assessed using 5-bromo-2-deoxyuridine incorporation. Apoptosis and cell-cycle distribution were analysed by flow cytometry; cyclin B1, Cdk1, p-Cdk1 Cdc25C, p-Cdc25C and p21 expressions were analysed by western blotting. mRNA levels were analysed by real time RT-PCR. RESULTS: Nadroparin inhibited cell proliferation by 30% in both cell lines; it affected the cell cycle in A549, but not in CALU-1 cells, inducing arrest in the G(2) /M phase. Nadroparin in A549 culture inhibited cyclin B1, Cdk1, Cdc25C and p-Cdc25C, while levels of p-Cdk1 were elevated; p21 expression was not altered. Dalteparin caused a similar reduction in A549 cell population growth; however, it did not alter cyclin B1 expression as expected, based on previous reports. Fondaparinux caused minimal inhibition of A549 cell population growth and no effect on either cell cycle or cyclin B1 expression. CONCLUSIONS: Nadroparin inhibited proliferation of A549 cells by inducing G(2) /M phase cell-cycle arrest that was dependent on the Cdc25C pathway, whereas CALU-1 cell proliferation was halted by as yet not elucidated modes.

Increased muscle expression of interleukin-17 in Duchenne muscular dystrophy.

OBJECTIVES: Duchenne muscular dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. Dystrophic muscle is characterized by chronic inflammation, and inflammatory mediators could be promising targets for innovative therapeutic interventions. We analyzed muscle biopsy samples of DMD-affected children to characterize interleukin (IL)-17 and Forkhead box P3 (Foxp3) expression levels and to identify possible correlations with clinical status. METHODS: Expression levels of IL-17, Foxp3, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), IL-6, and transforming growth factor-ß (TGF-ß) were analyzed by real-time PCR in muscle biopsy samples from patients with DMD (n = 27) and juvenile dermatomyositis (JDM) (n = 8). Motor outcome of patients with DMD was evaluated by North Star Ambulatory Assessment score. RESULTS: In DMD, we found higher levels of IL-17 and lower levels of Foxp3 mRNA compared with those for a typical inflammatory myopathy, JDM. Moreover, the IL-17/Foxp3 ratio was higher in DMD than in JDM biopsy samples. IL-17 mRNA levels appeared to be related to the expression levels of other proinflammatory cytokines (TNF-α and MCP-1) and significantly associated with clinical outcome of patients. CONCLUSIONS: The association of IL-17 expression with levels of other inflammatory cytokines and with the clinical course of DMD suggests a possible pathogenic role of IL-17.

Evaluation of safety and efficacy of DNA vaccines against bovine herpesvirus-1 (BoHV-1) in calves.

Four DNA vaccines against BoHV-1 were evaluated for their efficacy in calves. Twelve animals were divided into four groups which were injected with four different DNA vaccines: pVAX-tgD (Vaccine A); pVAX-tgD co-immunised with pVAX-48CpG (Vaccine B); pVAX-UbiLacI-tgD-L (Vaccine C); pVAX-UbiLacI-tgD-L co-immunised with pVAX-48CpG (Vaccine D). Three additional calves were given the plasmid vector and served as controls. Ninety days after the first vaccination all calves were challenge infected with BoHV-1. All animals developed a severe form of infections bovine rhinotracheitis. Only the calves given the pVAX-tgD co-immunised with pVAX-48CpG (Vaccine B) developed humoral antibodies against BoHV-1 between 56 and 90 days after the first vaccination, whereas in calves of other groups and in the controls, antibodies appeared only after the infection. In the calves vaccinated with either pVAX-tgD (Vaccine A) or pVAX-tgD combined with pVAX-48CpG (Vaccine B), BoHV-1-specific IFN-γ secreting cells were detected in PBMCs 90 days after the first vaccination and their number increased after challenge exposure. In the other groups the IFN-γ secreting cells were detected after virus infection and at low values.

Pathogenesis and subsequent cross-protection of influenza virus infection in pigs sustained by an H1N2 strain.

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co-circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross-protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre-determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross-protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post-challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross-protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post-infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross-protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub-type induced less protection against re-infection by homologous and heterologous virus than H1N1 sub-type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross-protection against heterologous infection.

From the fetal liver to spleen and gut: the highway to natural antibody.

The film of sIgA lining the intestinal epithelium plays a role in the regulation of the commensal microflora and prevention of pathogen invasion. We show that, in the absence of intentional immunization, all sIgA in the gut is produced by B-1a B cells. We also show that B-1a B cells and sIgA derive from lineage-negative precursors found in the fetal liver and located in the spleen after birth. The splenic precursors do not generate B cells of the adaptive immune system in bone marrow, spleen, and lymph nodes, but efficiently replenish the cells producing the natural antibodies. Therefore, B-1a B cells with their splenic progenitors and their progeny of plasma cells fill the same function of the primordial immune system of lower vertebrates. The natural antibodies in the serum and on the intestinal epithelium may be an evolutionary ancient tool for the immediate protection against commensal and pathogenic bacteria.

Expanding the clinical spectrum of POMT1 phenotype.

Mutations in POMT1 have been identified in Walker-Warburg syndrome and in patients with limb-girdle muscular dystrophy and mental retardation (LGMD2K). The authors report new POMT1 mutations in three unrelated children with severe motor impairment, leg hypertrophy, and mental retardation but without brain and ocular malformations. These patients are similar to LGMD2K but have earlier onset and more severe motor disability. The current findings expand the spectrum of POMT1-associated phenotypes.

Major myofibrillar changes in early onset myopathy due to de novo heterozygous missense mutation in lamin A/C gene.

Mutations in the lamin A/C gene (LMNA) have been associated with neuromuscular diseases and more complex syndromes, involving bone and adipose tissue. We report on a case of early onset myopathy due to a heterozygous LMNA mutation in exon 9, characterized by the presence of a marked number of cytoplasmic bodies with extensive myofibrillar abnormalities and Z-disk disruption in skeletal muscle. This case suggests there is a need to increase the list of genes to be screened in patients with myofibrillar myopathy.

Phenotypic heterogeneity in two unrelated Danon patients associated with the same LAMP-2 gene mutation.

Danon disease, an X-linked cardioskeletal myopathy caused by primary deficiency of lysosome-associated membrane protein-2 (LAMP-2), is clinically characterized by cardiomyopathy, myopathy, and variable mental retardation. The pathological hallmark of the disease is the absence of LAMP-2 immunohistochemical staining in muscle. The LAMP-2 gene mutations reported thus far are generally private mutations. We describe two cases of Danon disease with different clinical presentation, in whom we identified the same exon skipping mutation c.928G>A in the LAMP-2 gene. The first patient was affected by an early onset myopathy and hypertrophic cardiomyopathy (HCM) that partially improved with drug treatment. A first muscle biopsy at age 4 months showed markedly increased glycogen, and acid maltase deficiency was ruled out biochemically. A second muscle biopsy, performed at age 3(1/2) years, showed very mild abnormalities. The second child at age 15 years had mild, diffuse muscle weakness and wasting, moderate mental deficiency, and HCM. Two serial biopsies performed at age 8 and 15 years showed similar findings of multiple esterase-positive vacuoles in type I myofibers. In both patients the immunohistochemical study demonstrated the absence of LAMP-2 in skeletal muscle.

The effect of volatile anesthetics on respiratory system resistance in patients with chronic obstructive pulmonary disease.

We examined the effect of isoflurane and sevoflurane on respiratory system resistance (Rmin,rs) in patients with chronic obstructive pulmonary disease (COPD). The diagnosis of COPD rests on the presence of airway obstruction, which is only partially reversible after bronchodilator treatment. Ninety-six consecutive patients undergoing thoracic surgery for peripheral lung cancer were enrolled. They were divided into two groups: preoperative forced expiratory volume in 1 s/forced vital capacity ratio <70% or >70%. Rmin,rs was measured after 5 and 10 min of maintenance anesthesia by using the constant flow/rapid occlusion method. Maintenance of anesthesia was randomized to thiopental 0.30 mg . kg(-1) . min(-1) or 1.1 minimum alveolar anesthetic concentration end-tidal isoflurane or sevoflurane. Eleven patients were excluded: two because anesthesia was erroneously induced with propofol and nine because of an incorrect tube position. Maintenance with thiopental failed to decrease Rmin,rs, whereas both volatile anesthetics were able to decrease Rmin,rs in patients with COPD. The percentage of patients who did not respond to volatile anesthetics was larger in those with COPD as well. In conclusion, we have demonstrated that isoflurane and sevoflurane produce bronchodilation in patients with COPD.

A review of HIV-1 Tat protein biological effects.

The authors have reviewed some biological properties of HIV-1 Tat protein, and have also reported some personal data. This viral regulatory protein is endowed with multifunctional activities, acting as an endogenous factor in the infected cells and exogenously, on those uninfected. In particular, Tat-induced proliferation and differentiation of HIV target cells which promotes viral infection, is discussed in this review. However, exogenous Tat protein can sometimes also produce, directly or indirectly, damaging effects in different organs and host systems, such as myocardium, kidney, liver and central nervous system (CNS). For example some data also demonstrate an increase in the apoptotic index induced by Tat at various levels, including the immune system. The effective role of HIV-1 Tat protein in promoting viral replication and its high immunogenicity suggest useful employment of this protein for therapeutic or preventive vaccine preparations.

A review of cardiovascular complications accompanying AIDS.

Manifestations of cardiovascular system involvement are not uncommon complications of HIV infection, especially in AIDS patients. However, the frequency of these manifestations is influenced by different variables including: survival prolongation in HIV-infected patients, because of advances in antiretroviral treatment; improvement of immunodepression and reduction in the occurrence of opportunistic infections; adverse effects of some drugs. At present, on the whole cardiovascular complications that are HIV correlated in the western world, including Italy, occur less frequently than in the past. However complications associated with alterations in lipometabolism prevail because they can be promoted by some protease inhibitors in predisposed subjects. The most frequently reported questions and a careful analysis of recent data in the medical literature regarding the most common HIV-correlated cardiovascular complications are discussed in this review.

Immunization against bovine herpesvirus-1 infection. Preliminary tests in calves with a DNA vaccine.

A bovine herpesvirus-1 (BHV-1) vaccine expressing glycoprotein D, the form with the transmembrane anchor removed, was evaluated for inducing immunity in calves. The plasmid encoding gD of BHV-1 was injected three times to nine calves, using three animals for each of the following routes: intramuscularly (i.m.), intradermally (i.d.), or intranasally (i.n.). Three additional calves were given the plasmid vector only and served as unvaccinated controls. When calves were subjected to challenge infection with BHV-1, all vaccinated calves as well as the controls developed a typical severe form of infectious bovine rhinotracheitis. However, compared to the controls, the vaccinated calves showed earlier clearance of challenge virus. Moreover, the calves given the vaccine i.m. developed neutralizing antibody to BHV-1 between 21 and 42 days following the first injection of vaccine, whereas in calves vaccinated either i.d. or i.n., as well as the controls, antibody first appeared in their sera 14 days post-challenge infection.

Autosomal dominant external ophthalmoplegia and bipolar affective disorder associated with a mutation in the ANT1 gene.

The authors report on a family with dominantly inherited progressive external ophthalmoplegia and a diagnostic and statistical manual (fourth revised edition) diagnosis of bipolar psychiatric disorder in several members. Skeletal muscle biopsy from the proposita showed decreased cytochrome c oxidase staining, several ragged-red fibers, and multiple mtDNA deletions. The authors identified a missense mutation (leucine 98-->proline) in the adenine nucleotide translocator 1 gene. The presence of bipolar affective disorder expands the phenotype of adenine nucleotide translocator 1 allelic variants.

[Part-time work at a hospital: study of atypical workers]. / Il part time in un'Azienda Ospedaliera: studio sui lavoratori atipici.

According to act 626/1994, employers have the duty to inform and train workers and their representatives. The implementation of training activities requires the following points: planning the training progra according to the needs of the target population, use of the methods aimed at promoting learning and the adoption of safe behaviour, setting-up of evaluation tools. The disciplines of risk perception and communication and adult training may provide useful contribution in this frame. At the light of the preliminary experiences in this field, the importance of the following items for workers, workers representatives and employers is emphasized: probabilistic causality models, role of cognitive and emotional factors in the learning process, definition of carcinogenic according to national and internationals organisation, meaning of TLV with respect to carcinogenic exposure, interaction between carcinogens in the case of multiple exposition, risk evaluation, preventive measures, transfer of carcinogen risk from workplace to domestic environment, due to lack of compliance with basic hygienic rules such proper use of work clothes.