Measuring and Modelling Nonlinear Elasticity of Ex Vivo Mouse Muscles.

Elastography is a noninvasive imaging technique that provides information on soft tissue stiffness. Young's modulus is typically used to characterize soft tissues' response to the applied force, as soft tissues are often considered linear elastic, isotropic, and quasi-incompressible materials. This approximation is reasonable for small strains, but soft tissues undergo large deformations also for small values of force and exhibit nonlinear elastic behavior. Outside the linear regime, the elastic modulus is dependent on the strain level and is different for any kind of tissue. The aim of this study was to characterize, ex vivo, the mechanical response of two different mice muscles to an external force. A system for transverse force-controlled uniaxial compression enabled obtaining the stress-strain (σ-ε) curve of the samples. The strain-dependent Young's modulus (SYM) model was adopted to reproduce muscle compression behavior and to predict the elastic modulus for large deformations. After that, a recursive linear model was employed to identify the initial linear region of the σ-ε curve. Results showed that both muscle types exhibited a strain hardening effect and that the SYM model provided good fitting of the entire σ-ε curves. The application of the recursive linear model allowed capturing the initial linear region in which the approximation of these tissues as linear elastic materials is reasonable. The residual analysis displayed that even if the SYM model better summarizes the muscle behavior on the entire region, the linear model is more precise when considering only the initial part of the σ-ε curve.

Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy.

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease. RESEARCH IN CONTEXT: Duchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a "cure" for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. In this context, we previously showed that genetic ablation of Protein Kinase C θ (PKCθ), an enzyme known to be involved in immune response, in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.

Congenital rubella still a public health problem in Italy: analysis of national surveillance data from 2005 to 2013.

In accordance with the goal of the World Health Organization Regional Office for Europe, the Italian national measles and rubella elimination plan aims to reduce the incidence of congenital rubella cases to less than one case per 100,000 live births by the end of 2015. We report national surveillance data for congenital rubella and rubella in pregnancy from 2005 to 2013. A total of 75 congenital rubella infections were reported; the national annual mean incidence was 1.5/100,000 live births, including probable and confirmed cases according to European Union case definition. Two peaks occurred in 2008 and 2012 (5.0 and 3.6/100,000 respectively). Overall, 160 rubella infections in pregnancy were reported; 69/148 women were multiparous and 38/126 had had a rubella antibody test before pregnancy. Among reported cases, there were 62 infected newborns, 31 voluntary abortions, one stillbirth and one spontaneous abortion. A total of 24 newborns were unclassified and 14 women were lost to follow-up, so underestimation is likely. To improve follow-up of cases, systematic procedures for monitoring infected mothers and children were introduced in 2013. To prevent congenital rubella, antibody screening before pregnancy and vaccination of susceptible women, including post-partum and post-abortum vaccination, should be promoted. Population coverage of two doses of measles-mumps-rubella vaccination of ≥ 95% should be maintained and knowledge of health professionals improved.

Epidemiology of pertussis in Italy: disease trends over the last century.

We reviewed the epidemiology of pertussis in Italy over the last 125 years to identify disease trends and factors that could have influenced these trends. We described mortality rates (1888-2012), case fatality rates (1925-2012), cumulative incidence rates (1925-2013) and age-specific incidence rates (1974-2013). We compared data from routine surveillance with data from a paediatric sentinel surveillance system to estimate under-notification. Pertussis mortality decreased from 42.5 per 100,000 population in 1890 to no reported pertussis-related death after 2002. Incidence decreased from 86.3 per 100,000 in 1927 to 1 per 100,000 after 2008. Vaccine coverage increased from 32.8% in 1993 to about 96% after 2006. As for under-notification, mean sentinel/routine surveillance incidence ratio increased with age (from 1.8 in <1 year-olds to 12.9 in 10-14 year-olds). Pertussis mortality decreased before the introduction of immunisation. Incidence has decreased only after the introduction of pertussis vaccine and in particular after the achievement of a high immunisation coverage with acellular vaccines. Routine surveillance does not show an increase in cumulative incidence nor in ≥ 15 year-olds as reported by other countries. Underrecognition because of atypical presentation and the infrequent use of laboratory tests may be responsible for under-notification, and therefore affect incidence reports and management of immunisation programmes.

Trends of human brucellosis in Italy, 1998-2010.

SUMMARY: We describe the epidemiological trends and spatial distribution of human brucellosis in Italy over 13 years (1998-2010). In the study period 8483 cases were notified in Italy, with a relevant decrease (-89%) from 1998 to 2010. Most cases were notified in southern Italy (Campania, Apulia, Calabria, Sicily). In these regions we observed relevant differences in the risk of brucellosis at province level. Cases were distributed with a seasonal pattern, male patients represented 60% of the cases and no significant differences were observed between age groups. We modelled the underreporting rate that ranged between 2 and 21 (average 12·5). According to our estimates the true number of cases would have ranged from 41 821 to 155 324 providing a far more severe picture of human brucellosis in Italy than the one provided by the surveillance system.

Dystrophic tendon functionality is recovered by muscle-specific expression of insulin-like growth factor in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder of skeletal muscle, characterized by a steady muscle weakness. By using the animal model for DMD, the mdx mice, we have previously demonstrated that biomechanical properties of tendinous tissue are also significantly affected in this muscle pathology. Muscle specific over-expression of insulin like growth factor-1 (mIgf-1) is known to induce a partial recovery in muscle functionality, in particular increasing the muscle absolute force, but not the specific force. To test whether Igf-1 muscle specific over-expression helps the recovery also in tendinous tissue, mechanical and cellular evaluation of mdx and mdx:MLC/mIgf-1 mice tendons has been performed. Mechanical properties were investigated by measuring the viscoelastic response of the tissue, while cell viability was evaluated by molecular assays. An absolute recovery in the mechanical properties of EDL and TA tendons was observed through the measurement of tissue viscoelasticity for several different frequencies of interest. Moreover, when compared with tendons from dystrophic mdx animals, mdx:MLC/mIgf-1 specimens showed an almost complete recovery in the number of viable cells for both extensor digitorum longus (EDL) and tibialis anterior (TA) tendons. Of note, the partial recovery in muscle functionality and the full recovery in tendons response, suggests that mIgf-1 muscle specific over-expression exerts its effect on tendons either indirectly, improving the tendon viability and its functional properties as a consequence of the reduction of the hostile muscle dystrophic environment, or acting directly on the tendon tissue, as a paracrine trophic factor.

Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury.

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

Scriptaid effects on breast cancer cell lines.

In breast cancer tumor expression of estrogen receptors (ERs) is important as a marker of prognosis and mostly as a predictor of response to endocrine therapy. In fact, the loss of α-ER expression leads to unresponsiveness to anti-hormone treatment. In a significant fraction of breast cancers, this loss of expression is a result of epigenetic mechanisms, such as DNA methylation and histone deacetylation, within the α-ER promoter. Previous studies have shown that pharmacologic inhibition of these mechanisms using the DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (AZA), and the histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), results in expression of functional α-ER mRNA and protein. Moreover, the activity of a novel HDAC inhibitor, Scriptaid, has been shown to induce inhibition of tumor growth in breast cancer and to cause re-expression of functional α-ER in α-ER negative breast cancer cells. We sought to better characterize the effects of Scriptaid on cell growth, apoptosis, and α-ER expression in α-ER-positive (MCF-7), α-ER-negative (MDA-MB-231), and α-ER-negative/Her-2 over-expressing (SKBr-3) human breast cancer cell lines. In all of these cell lines Scriptaid treatment resulted in significant growth inhibition and apoptosis, and RT-PCR confirmed an increase of α-ER mRNA transcript in MDA-MB-231 after 48 h of Scriptaid treatment. Furthermore, following treatment with Scriptaid, the formerly unresponsive MDA-MB-231 and SKBr-3 breast cancer cells became responsive to tamoxifen. These results show that the HDAC inhibitor Scriptaid is able to sensitize tamoxifen hormone-resistant breast cancer cells, and that Scriptaid or related HDAC inhibitors are candidates for further study in breast cancer.

Measuring tendon properties in mdx mice: cell viability and viscoelastic characteristics.

Muscular dystrophy is a genetic disorder of skeletal muscle characterized by progressive muscle weakness. Here we assessed whether muscle wasting affects cell viability and mechanical properties of extensor digitorum longus (EDL) and of tibialis anterior (TA) tendons from mdx dystrophic mice compared to wild type (WT) mice. mdx mice represent the classical animal model for human Duchenne muscular dystrophy, and show several signs of the pathology, including a decrease in specific force and an increase of fibrotic index. Cell viability of tendons was evaluated by histological analysis, and viscoelastic properties have been assessed by a rapid measurement protocol that allowed us to compute, at the same time, tissue complex compliance for all the frequencies of interest. Confocal microscopy and mechanical properties measurements revealed that mdx tendons, compared to WT ones, have an increase in the number of dead cells and a significant reduction in tissue elasticity for all the frequencies that were tested. These findings indicate a reduced quality of the tissue. Moreover, mdx tendons have an increase in the viscous response, indicating that during dynamic loading, they dissipate more energy compared to WT. Our results demonstrate that muscular dystrophy involves not only muscle wasting, but also alteration in the viscoelastic properties of tendons, suggesting a paracrine effect of altered skeletal muscle on tendinous tissue.

Effectiveness of influenza vaccination: a survey within the Italian Ministry of Health personnel.

An initiative of active prevention, to promote an influenza vaccination programme, has been carried out in the Ministry of Health of Rome, Directorate General of Health Prevention: during October 2004 influenza vaccine has been actively offered to all the employees; 46 out of the 129 workers (about 36% of the total) accepted to be vaccinated. Afterward, the propensity to get influenza of the vaccinated has been compared with that of a control group, operating in the same setting, but not vaccinated. Taking into account that the groups in question is far too small, the statistical results showed that influenza vaccination of healthy working adults did not reduce the rates of influenza-like-illness (ILI), in addition, the difference between average duration of disease in unvaccinated persons was not statistically significant.